Development System Testing virus region 4 (VR4) mutant cross-species compatible Adeno Associated Viruses (ccAAVs) in mice. | SCGE Toolkit C57BL/6 mice (N=3) were injected intravenously at a dose of 5e13 vg/kg per mouse with a self-complementary AAV9 or ccAAV vector encoding an mCherry reporter. The biodistribution of of virus transduction was chacterized in various tissues and cell types by fluorescence imaging quantification.
Experiment: Testing virus region 4 (VR4) mutant cross-species compatible Adeno Associated Viruses (ccAAVs) in mice.
PI:
Aravind Asokan, PhD
Description: C57BL/6 mice (N=3) were injected intravenously at a dose of 5e13 vg/kg per mouse with a self-complementary AAV9 or ccAAV vector encoding an mCherry reporter. The biodistribution of of virus transduction was chacterized in various tissues and cell types by fluorescence imaging quantification.
Delivery Assays:
Native fluorescence was quantified using Image J. For each animal, 6 images were taken from 2 sections and used to calculate corrected total cell fluorescence using the formula: Integrated Density – (Area of selected image X Mean fluorescence of background readings).
Relative neuronal transduction levels were quantified by counting the number of transduced neurons, identified based on morphology, for each brain region per 50um sagittal section
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Results
SCGE Toolkit downloaded on: 2024/11/21 11:09:13; Please cite the Somatic Cell Genome Editing Consortium Toolkit NIH HG010423 when using publicly accessible data in formal presentation or publication. SCGE Experment ID: 18000000003. PI:
Aravind Asokan PhD
Cross-species evolution of a highly potent AAV variant for therapeutic gene transfer and genome editing. NCBI
Expression of VR4 ccAAVs in the brain following IV injection
Expression of VR4 ccAAVs in the brain. Adult C57/B6 mice were injected intravenously at 5e13 vg/kg (N=3). A self-complementary Mcherry cassette was packaged into lead ccAAV mutants driven by a chicken-beta actin human hybrid (CBh) promoter. Mice were sacrificed 4-weeks post injection brain was post-fixed in 4% PFA for 24 hrs. Tissue was cut into 50mm sagittal sections using a vibratome. Immunohistochemical staining of brain sections revealed neuronal and astrocytic expression (representative images are shown).
Expression of VR4 ccAAVs in cardiac and skeletal muscle
Expression of VR4 ccAAVs in cardiac and skeletal muscle. Adult C57/B6 mice were injected intravenously at 5e13 vg/kg (N=3). A self-complementary Mcherry cassette was packaged into lead ccAAV mutants driven by a chicken-beta actin human hybrid (CBh) promoter. Mice were sacrificed 4-weeks post injection and heart and skeletal muscle was post-fixed in 4% PFA for 24 hrs. Fixed tissue was embedded in agarose and cut into 50mm thick sections using a vibratome. Native fluorescence was quantified using Image J. For each animal, 6 images were taken from 2 sections and used to calculate corrected total cell fluorescence using the formula: Integrated Density (Area of selected image X Mean fluorescence of background readings).
Expression of VR4 ccAAVs in liver and kidney
Expression of VR4 ccAAVs in liver and kidney. Adult C57/B6 mice were injected intravenously at 5e13 vg/kg (N=3). A self-complementary Mcherry cassette was packaged into lead ccAAV mutants driven by a chicken-beta actin human hybrid (CBh) promoter. Mice were sacrificed 4-weeks post injection and liver and kidney was post-fixed in 4% PFA for 24 hrs. Fixed tissue was embedded in agarose and cut into 50mm thick sections using a vibratome. Native fluorescence was quantified using Image J. For each animal, 6 images were taken from 2 sections and used to calculate corrected total cell fluorescence using the formula: Integrated Density (Area of selected image X Mean fluorescence of background readings).
