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: Glia

Article Title: A Modified Flavonoid Accelerates Oligodendrocyte Maturation and Functional Remyelination

doi: 10.1002/glia.23715

Figure Lengend Snippet: Effects of the S3 flavonoid on the activities of different hyaluronidases. (A) Effects of 20 μM S3 (“+”) on HA digestion by BTH, recombinant Hyal1, recombinant PH20 (rPH20), and Streptomyces hyaluronidase compared to enzymes treated with vehicle alone (“-“). The “HA” lane was incubated without S3 but with the vehicle from the hyaluronidases. (B) Effects of the S3 flavonoid on HA digestion by lysates from HEK-293 cells transfected with empty vector (“vector”) or cells transfected with Hyal1, Hyal2, or PH20 compared to HA digested by rPH20 or BTH or to undigested HA “HA”. (C) Effects of different concentrations of S3 or vehicle alone (DMSO - “D”) on HA digestion by HEK-293 cells either untransfected (“untrans”), transfected with vector alone, or transfected with an expression vector carrying the cDNA for Cemip. (D) Quantification of the inhibition of HA digestion by S3 (20 μM) in the presence of recombinant Hyal1, recombinant PH20, BTH, or Streptomyces hyaluronidase. *p<0.01. (E) Quantification of HA digestion in live cell cultures of HEK-293 cells treated with S3 (20 μM) following transfection with either Hyal1, Hyal2, PH20 or Cemip expression vectors. *p<0.005.

Article Snippet: Recombinant bovine SPAM1 (rPH20, 1.5 μg/ml, R&D Systems), Recombinant Human Hyaluronidase HYAL1 (rhHyal1, 6 μg/ml, R&D Systems), recombinant Human protein HYAL2 (rhHyal2, 40 μg/ml, MybioSource), Streptomyces hyaluronidase (StrepH; 7.5U/ml, Sigma), and bovine testicular hyaluronidase (BTH; Sigma, 10–20U/ml) were diluted in pH 3.5 or 4.5 (sodium citrate buffer) or pH 7.0 (phosphate-buffered saline, PBS) sample buffer.

Techniques: Recombinant, Incubation, Transfection, Plasmid Preparation, Expressing, Inhibition

Journal: Glia

Article Title: A Modified Flavonoid Accelerates Oligodendrocyte Maturation and Functional Remyelination

doi: 10.1002/glia.23715

Figure Lengend Snippet: Effects of blocking hyaluronidase activity on HA-mediated inhibition of OPC maturation. (A) Mouse OPCs grown under conditions that favor maturation in the absence of HA. (B) OPCs grown as in A in the presence of HMW HA. (C) OPCs grown as in B in the presence of 2 μM S3. (D) OPCs grown as in C in the presence of 25 U rPH20. (E) OPCs grown as in B in the presence of 25 μM Vcpal. Red = MBP; green = PDGFRα. Scale bars = 50 μm. (F) Quantification of the results in A-E. c = vehicle control; vc = Vcpal; P = PH20. Note that the data to the right of the dashed lines was from a separate experiment where HA was not added to the cultures. *p<0.001; **p<0.0005

Article Snippet: Recombinant bovine SPAM1 (rPH20, 1.5 μg/ml, R&D Systems), Recombinant Human Hyaluronidase HYAL1 (rhHyal1, 6 μg/ml, R&D Systems), recombinant Human protein HYAL2 (rhHyal2, 40 μg/ml, MybioSource), Streptomyces hyaluronidase (StrepH; 7.5U/ml, Sigma), and bovine testicular hyaluronidase (BTH; Sigma, 10–20U/ml) were diluted in pH 3.5 or 4.5 (sodium citrate buffer) or pH 7.0 (phosphate-buffered saline, PBS) sample buffer.

Techniques: Blocking Assay, Activity Assay, Inhibition, Control