Journal: bioRxiv

Article Title: Preclinical studies for plant-based oral enzyme replacement therapy (Oral-ERT) in Pompe disease knockout mice with transgenic tobacco seeds expressing human GAA (tobrhGAA)

doi: 10.1101/2021.11.11.468227

Figure Lengend Snippet: A human PD myoblast cell line was exposed to equivalent amounts of a lysate of tobrhGAA or an rhGAA for 48 hr and assayed for GAA and NAG. We found that tobrhGAA increased GAA to 24-35% of normal (mean±SD).

Article Snippet: Mock treated GAA and normal myoblast cells were controls plus cells treated with equivalent amounts of a rhGAA (R&D Systems #8329-GH-025).

Techniques:

Journal: Human Gene Therapy

Article Title: Efficacy and Safety of a Krabbe Disease Gene Therapy

doi: 10.1089/hum.2021.245

Figure Lengend Snippet: Proof-of-concept efficacy in neonatal Twitcher mice. (A) Survival curve of Twitcher mice injected intravenously on PND0 with 50 μL of PBS (vehicle, n = 8; median survival 40.5 days) or with 1 × 10 11 GC of AAVhu68.CB7.hGALCco.rBG diluted in 50 μL PBS ( n = 6; median survival 49 days). *** p < 0.001 Log-rank (Mantel-Cox) test. (B) Survival curve of Twitcher mice injected in the lateral cerebral ventricle (ICV) on PND0 with 2 μL of PBS (vehicle, n = 8; median survival 43 days), with 2 × 10 10 GC of AAVhu68.CB7.hGALCco.rBG in 2 μL PBS (low dose, n = 10; median survival 62 days), with 5 × 10 10 GC of AAVhu68.CB7.hGALCco.rBG in 2 μL PBS (mid dose, n = 12; median survival 99 days), or with 1 × 10 11 GC of AAVhu68.CB7.hGALCco.rBG in 2 × 2 μL PBS (bilateral ICV, high dose, n = 12; median survival 130 days). **** p < 0.0001 Log-rank (Mantel-Cox) test. (C) Neuromotor function assessed by the accelerated rotarod test on PND35 in mice treated via neonatal ICV administration [same animals as in (B) ]. ** p < 0.01, *** p < 0.001, **** p < 0.0001, Kruskal-Wallis test followed by post hoc Dunn's multiple comparison test, alpha = 0.05, comparison to vehicle Twitcher mice. (D) GALC enzyme activity in brain lysate from tissue obtained at humane endpoint in animals administered IV or ICV [same animals as in (A, B) ]. * p < 0.05, **** p < 0.0001, Kruskal-Wallis test followed by post hoc Dunn's multiple comparison test, alpha = 0.05, comparison to vehicle Twitcher mice. GALC, galactosylceramidase; GC, genome copies; ICV, intracerebroventricular; IV, intravenous; PBS, phosphate-buffered saline; PND, postnatal day.

Article Snippet: We tested the sensitivity and specificity of the kit using recombinant human GALC (7310-GH; R&D Systems) and recombinant human beta-galactosidase (6464-GH; R&D Systems).

Techniques: Injection, Comparison, Activity Assay, Saline

Journal: Human Gene Therapy

Article Title: Efficacy and Safety of a Krabbe Disease Gene Therapy

doi: 10.1089/hum.2021.245

Figure Lengend Snippet: MED study in juvenile postdisease onset Twitcher mice. (A) Compound clinical severity score in Twitcher mice treated on PND12–14 ICV with either 4 μL of artificial CSF (vehicle, n = 17), or 4 μL of AAVhu68.CB7.hGALCco.rBG at the following doses: 6.8 × 10 9 GC ( n = 16), 2 × 10 10 GC ( n = 17), 6.8 × 10 10 ( n = 17), or 2 × 10 11 GC ( n = 16). WT littermates were treated ICV with 4 μL of artificial CSF ( n = 17). The operator was blinded to the mice genotype and treatment. **** p < 0.0001 linear mixed-effect modeling comparing the clinical score change over time compared to the vehicle Twitcher group, alpha = 0.05. (B) Neuromotor function assessed by the accelerated rotarod test on PND35 in same mice as in (A) ** p < 0.01, *** p < 0.001, **** p < 0.0001, Kruskal-Wallis test followed by post hoc Dunn's multiple comparison test, alpha = 0.05, comparison to vehicle Twitcher mice. (C) GALC enzyme activity in brain (sagittal half), heart (sagittal half), and liver (half of the left lobe) lysate from tissue obtained either at scheduled necropsy (PND40, half of the animals) or humane endpoint (half of the animals) in the same animals as in (A) * p < 0.05, **** p < 0.0001, Kruskal-Wallis test followed by post hoc Dunn's multiple comparison test, alpha = 0.05, comparison to vehicle Twitcher mice. (D) Neuroinflammation quantification in the cerebral cortex, spinal cord, and sciatic nerve measured by mean object area of IBA1-positive cells stained by immunohistochemistry in tissues collected at the fixed necropsy time PND40. *** p < 0.001, **** p < 0.0001, Kruskal-Wallis test followed by post hoc Dunn's multiple comparison test, alpha = 0.05, comparison to vehicle-treated Twitcher mice. Representative images of IBA1 IHC from the vehicle groups (Twitcher and WT mice) and the HD group (2 × 10 11 GC, ICV PND12–14). CSF, cerebrospinal fluid; MED, minimum effective dose; WT, wild type.

Article Snippet: We tested the sensitivity and specificity of the kit using recombinant human GALC (7310-GH; R&D Systems) and recombinant human beta-galactosidase (6464-GH; R&D Systems).

Techniques: Comparison, Activity Assay, Staining, Immunohistochemistry

Journal: Human Gene Therapy

Article Title: Efficacy and Safety of a Krabbe Disease Gene Therapy

doi: 10.1089/hum.2021.245

Figure Lengend Snippet: ICM efficacy study in Krabbe dogs, nerve-conduction studies, CSF biomarkers, and MRI. (A) Nerve-conduction velocities in the radial (sensory), sciatic (motor), ulnar (motor), and tibial (motor) nerves in 2- to 3-week-old Krabbe dogs treated ICM with either (1) 1 mL of artificial CSF (vehicle, n = 2); or (2) 3 × 10 13 GC of AAVhu68.CB7.cGAMCco.rBG in 1 mL ( n = 4). A WT littermate that received 1 mL of artificial CSF ICM was used as a control. Two treated dogs were sacrificed at the scheduled timepoint of 6 months postinjection for tissue collection. Two treated dogs and the vehicle-treated Krabbe dogs were followed until humane endpoint. (B) Quantification of CSF psychosine and GalCer levels and CSF GALC enzyme activity. The dotted line represent the average GALC activity value from the serial CSF timepoints of the WT control (K928). (C) Brain MRI WM intensity semiquantitative scoring. (D) Brain MRI examples from one Krabbe vehicle dog (8 weeks), one Krabbe dog treated with AAV (10 weeks), and one WT control littermate (10 weeks). A dark hypointense WM represents normal myelination ( stars ); a grey isointense signal represents mild demyelination ( arrowhead ); a white hyperintense signal represents marked demyelination ( arrows ). AAV, adeno-associated virus; GalCer, galactosylceramide; ICM, intracisterna magna; MRI, magnetic resonance imaging; WM, white matter.

Article Snippet: We tested the sensitivity and specificity of the kit using recombinant human GALC (7310-GH; R&D Systems) and recombinant human beta-galactosidase (6464-GH; R&D Systems).

Techniques: Control, Activity Assay, Virus, Magnetic Resonance Imaging

Journal: Human Gene Therapy

Article Title: Efficacy and Safety of a Krabbe Disease Gene Therapy

doi: 10.1089/hum.2021.245

Figure Lengend Snippet: ICM efficacy study in Krabbe dogs, neuroinflammation and GALC levels. (A) Representative pictures of IBA1 staining by IHC illustrating the macrophage/microglial neuroinflammation in Krabbe dogs treated with vehicle control or with AAV ICM. (B) Quantification of the mean area of the IBA1-positive signal in different neuroanatomical regions of the brain, spinal cord, and sciatic nerve of Krabbe dogs treated with AAV or vehicle. Each bar represents an animal. Error bars when present (cerebral cortical WM, corpus callosum, centrum semiovale, and internal capsule), represent the standard deviation from quantification of multiple slides when the neuroanatomical region was present on more than one brain section. (C) GALC activity in tissue lysate (50 μg protein per reaction, 2 h incubation) relative to the WT control ( dotted line ). Each bar represents one animal. WM, white matter.

Article Snippet: We tested the sensitivity and specificity of the kit using recombinant human GALC (7310-GH; R&D Systems) and recombinant human beta-galactosidase (6464-GH; R&D Systems).

Techniques: Staining, Control, Standard Deviation, Activity Assay, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Quantification profiles of enzyme activity, secretion, and psychosine levels of Krabbe disease galactosylceramidase missense variants

doi: 10.1016/j.jbc.2025.110315

Figure Lengend Snippet: Generation and validation of MO3.13/ GALC -KO cells. A , human GALC gene sequences ( red ) adjacent to PAM sites ( bolded ) are targeted for Cas9-mediated cleavage by the sgRNA vectors. A downstream nonsense mutation ( asterisk ) is introduced upon successful targeted deletion (highlighted yellow ). B , confirmation of targeted deletion of the 85 bp region in the KO cell line by PCR amplification, compared to control WT cells. C , Western blot analysis of GALC and GAPDH proteins in native MO3.13 cells (WT), GALC-KO cells (KO), and GALC-overexpressing cells (OE). D , GALC activity and ( E ) psychosine levels in WT and KO cells. Statistical significance is determined using an unpaired t test (two-tailed, 3–4 independent experimental replicates, 95% confidence interval, ∗∗ p < 0.01, ∗∗∗ p < 0.001). PAM, protospacer adjacent motif.

Article Snippet: Standard curve samples were prepared by diluting recombinant human GALC protein (R&D Systems) in culture supernatant from KO cells to 0.8, 1.6, 3.2, 6.3, 12.5, 25, and 50 ng/ml.

Techniques: Biomarker Discovery, Mutagenesis, Amplification, Control, Western Blot, Activity Assay, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Quantification profiles of enzyme activity, secretion, and psychosine levels of Krabbe disease galactosylceramidase missense variants

doi: 10.1016/j.jbc.2025.110315

Figure Lengend Snippet: Location and distribution of KD-related MMVs on the human GALC protein. The schematic diagram shows the distribution of clinically relevant MMVs on the human GALC protein. Human Genome Variation Society (HGVS) nomenclature is applied. The main structural domains of GALC are indicated: signal peptide (SP) (1–42 a.a.), TIM barrel domain (57–353 a.a.), β-sandwich domain (354–468 a.a.), and lectin-binding domain (488–685 a.a). Key residues involved in catalytic function and substrate-binding are labeled in red . Polymorphic variants are labeled in blue . Active site variants are in bolded font . KD, Krabbe disease; MMV, missense mutation variant; TIM, triosephosphate isomerase.

Article Snippet: Standard curve samples were prepared by diluting recombinant human GALC protein (R&D Systems) in culture supernatant from KO cells to 0.8, 1.6, 3.2, 6.3, 12.5, 25, and 50 ng/ml.

Techniques: Binding Assay, Labeling, Mutagenesis, Variant Assay

Journal: The Journal of Biological Chemistry

Article Title: Quantification profiles of enzyme activity, secretion, and psychosine levels of Krabbe disease galactosylceramidase missense variants

doi: 10.1016/j.jbc.2025.110315

Figure Lengend Snippet: Accumulation of pre-GALC protein in GALC MMV cell models. A , intracellular pre-GALC and GAPDH proteins were detected by Western blot. The bottom two blots show MMVs in the absence (−) and presence (+) of the p.I562T polymorphic background. The same GAPDH blots were used to normalize both pre-GALC ( current panel ) and lys-GALC protein levels ( , A – D ), as pre-GALC and lys-GALC were detected on the same blot but at different molecular weights. B , pre-GALC levels (normalized to GAPDH) detected by Western blot are significantly correlated with pre-GALC levels measured by sandwich ELISA in the MMV cell models (Pearson r = 0.71, p < 0.0001, n = 35). C , pre-GALC levels measured by ELISA plotted against the structural location of the MMVs, categorized by their position on the TIM barrel ( red ), β-sandwich ( green ) and lectin-binding ( blue ) domains. Ten out of 22 MMVs significantly increase pre-GALC levels compared with WT-GALC in the MMV cell models. (Unpaired t test, two-tailed, three independent experimental replicates, 95% confidence interval; ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001). MMV, missense mutation variant; TIM, triosephosphate isomerase.

Article Snippet: Standard curve samples were prepared by diluting recombinant human GALC protein (R&D Systems) in culture supernatant from KO cells to 0.8, 1.6, 3.2, 6.3, 12.5, 25, and 50 ng/ml.

Techniques: Western Blot, Sandwich ELISA, Enzyme-linked Immunosorbent Assay, Binding Assay, Two Tailed Test, Mutagenesis, Variant Assay

Journal: The Journal of Biological Chemistry

Article Title: Quantification profiles of enzyme activity, secretion, and psychosine levels of Krabbe disease galactosylceramidase missense variants

doi: 10.1016/j.jbc.2025.110315

Figure Lengend Snippet: GALC activity correlates with sec-GALC levels in the MMV cell models. Correlations among GALC activity, sec-GALC levels, and pre-GALC protein levels in the GALC MMV expressing cells were analyzed using the Pearson correlation method. A , GALC activity is significantly correlated with sec-GALC levels in the cell models (Pearson r = 0.5, p < 0.01, n = 37). No significant correlations are found between ( B ) GALC activity and pre-GALC levels, and between ( C ) sec-GALC and pre-GALC levels. MMV, missense mutation variant.

Article Snippet: Standard curve samples were prepared by diluting recombinant human GALC protein (R&D Systems) in culture supernatant from KO cells to 0.8, 1.6, 3.2, 6.3, 12.5, 25, and 50 ng/ml.

Techniques: Activity Assay, Expressing, Mutagenesis, Variant Assay