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In Vivo
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AAV Tropism project
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Ten AAV serotypes delivering Cre recombinase were tested by intravenous delivery into Ai9 mice and chacterized for biodistribution across 20 tissues by quantitative PCR and imaging |
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2023-02-10
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In Vitro
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AAV tropism in kidney organoids cultured under flow
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Human kidney organoids generated from human ES and iPS cells are used to evaluate tropism of AAV2, AAV8, and AAV9 that express eGFP under CMV promoter. Organoids are exposed to AAVs at MOI 10^5 from differentiation day 21 to 28. Four culture conditions were tested: U-well suspension culture, static culture on gelbrin, low flow on a chip coated with gelbrin, and high flow on a chip coated with gelbrin. Immunohistochemistry was performed to visualize transduced cells with staining for podocytes (PODXL) and proximal tubules (LTL). AAV2/2 shows the highest transduction efficiency among three serotypes evaluated, and the GFP expression is highest in the static condition. |
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2023-09-22
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In Vitro
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AAV tropism in kidney organoids derived from human pluripotent stem cells.
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Human kidney organoids generated from human ES and iPS cells are used to evaluate tropism of AAV2, AAV8, and AAV9 that express eGFP under CMV promoter. Organoids are exposed to AAVs at MOI 10^5 from differentiaiton day 21 to 28. Immunohistochemistry is performed to visualize transduced cells with staining for podocytes (PODXL), proximal tubules (LTL), loops of Henle and distal nephrons (CDH1), interstitial stromal cells (PDGFRb), and endothelia (CD31). AAV2/2 shows the highest transduction efficiency among three serotypes evaluated, and the GFP expression is highest in proximal tubular cells. |
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2023-09-22
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In Vivo
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[Validation]
Independent validation for Asokan Delivery Team: Evolving High Potency AAV Vectors for Neuromuscular Genome Editing.
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Quantification of CRISPR/Cas editing in liver and heart following custom AAV-mediated delivery. Detection of editing in non-target tissues. |
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2021-03-30
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In Vivo
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On-target editing compared to 14 circle-seq nominated off-target sites of adenine base editor delivered by BE-eVLP vs AAV in the C57BL/6 liver
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On-target editing compared to off-target editing at 14 CIRCLE seq nominated sites in livers of an adenine base editor delivered by engineered virus-like particles (BE-eVLPs). Treated mice vs. untreated vs. AAV was assessed one week after systemic administration of BE-eVLPs or AAV-Pcsk9 to C57BL/6 mice. DNA sequencing reads containing A-T to G-C mutations within protospacer positions 4-10. |
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2022-04-15
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In Vitro
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AAV2/2 transduction efficiencies in kidney organoids cultured under flow determined by FACS
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Human kidney orgnaoids generated from human ES and iPS cells are used to evaluate tropism of AAV2 that expresses eGFP under CMV promoter. Organoids are exposed to AAVs at MOI 10^5 from differentiation day 22 to 23. Four culture conditions are tested: full flow on a chip, pulsed flow on a chip, no flow on a chip, U-well suspension culture. Flow cytometry is performed to quantify the transduction efficiency in LTL+ proximal tubules. U-well culture shows the highest transduction efficiency among those four conditions. |
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2023-09-22
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In Vitro
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A novel human T cell platform to define biological adverse effects of genome editing
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110 guide RNAs and SpCas9 were transfected into human T-cells. Indel rates were measured by targeted amplicon deep sequencing. |
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2020-12-09
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In Vivo
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Comparing CRISPR/Cas9 gene editing efficencies between AAV9 and AAVcc47 in Ai9 mice with a 1:1 cas9 to sgRNA ratio (CMV promoter)
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A dual vector strategy was employed: one delivering two single guide RNAs targeting the Rosa26 locus and one delivering CMV driven SaCas9 (both single stranded AAV cassettes). This strategy was evaluted with both AAV9 (n=3) and AAVcc47 (n=3) by intravenous injection in Ai9 mice. A total dose of 3e12vg was injected into each mouse (1.5e12vg each vector mixed 1:1) and organs were harvested 4 weeks post injection. Editing efficency was determined by calculating percent TdTomato+ cells normalized to Dapi+ cells in liver and heart. |
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2021-09-16
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In Vitro
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Testing AAV5 for activation of tdTomato in HEK293T cells
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AAV shuttle plasmids expressing SpCas9 and guide RNAs targeting the Ai9 transgene were tested in HEK293T cells by transient transfection. Both delivery and gene editing were detected by fluorescence. |
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2020-10-20
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In Vivo
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Testing AAV5 for activation of tdTomato in mouse airway
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AAV2/5 mediated gene editing in the mouse airway was tested by deliverying SpCas9 and guide RNAs targeting the Ai9 transgene in Ai9 transgenic mice. Viral delivery was detected by GFP expression and gene editing quantified by tdTomato activation |
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2020-10-20
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In Vivo
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Cre Recombinase dose escalation study in Ai9 mice
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A single stranded cmv cre cassette was packaged into AAV9 or AAVcc47 and injected intravenously in Ai9 mice. We injected n=3 at three different doses (1e10, 1e11, 1e12 vg) and harvested organs 4 weeks post injection. Fluorescence intensity in liver, heart, and skeletal muscle was quantified with tiff based images in Image J and neuronal transduction from each vector was quantified at the 1e12vg dose by counting the number of tdTomato+ neurons and number of NeuN+ cells from multiple sections and images. |
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2021-09-16
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In Vitro
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Delivery of saCas9 and gRNAs to nephron epithelia by AAV2 in kidney organoids.
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An AAV2 viral vector carrying SaCas9 and AAV2 vector expressing mCherry and carrying two gRNAs against the DMD gene were used in human kidney organoids to assess transgene delivery to nephron epithelia. Organoids were treated with both AAVs at MOI 10^5 for one week. Delivery of Cas9 and gRNA vectors to nephron cell types were evaluted by immunohistochemistry. Note: additional data not shown demonstrated mCherry and SaCas9 were not detectable in non-transduced cells. |
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2023-09-22
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In Vitro
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Biomarker assays to evaluate toxicity and inflammation induced by AAVs in ES cell derived kidney organoids.
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Human kidney organoids generated from human ES are used to evaluate toxicity and inflammation induced by AAV2, AAV8, and AAV9 that express eGFP under CMV promoter. Organoids are exposed to AAVs at MOI 10^5 . MCP-1 and KIM-1 levels were measured using Luminex (Bioplex 200). |
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2023-09-22
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In Vitro
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Biomarker assays to evaluate toxicity induced by AAVs in iPS cell derived kidney organoids.
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Human kidney organoids generated from human iPS cells are used to evaluate toxicity induced by AAV2, AAV8, and AAV9 that express eGFP under CMV promoter. Organoids are exposed to AAVs at MOI 10^5 . KIM-1 levels were measured using Luminex (Bioplex 200). |
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2023-09-22
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In Vivo
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Testing virus region 4 (VR4) mutant cross-species compatible Adeno Associated Viruses (ccAAVs) in mice.
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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. |
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2020-11-19
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In Vivo
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Testing gRNA sequence and gRNA scaffold modified in Ai9 mice.
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3e11 vg/mouse of AAV-BI28:GFAP-SaCas9-WPRE-pA and 3e11 vg/mouse of AAV-BI28:GFAP-NLS-GFP-U6-L1-U6-R2 were codelivered intravenously to adult male and female Ai9 mice. Editing was assessed in brain sections 4 weeks later. |
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2021-04-17
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In Vivo
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[Validation]
Independent validation of Deverman delivery platform using engineered AAVs to deliver CRSIPR/Cas9 to mouse brain
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Validation of delivery of AAV custom designed to cross the blood-brain barrier for CRISPR/Cas9 editing. Editing detected and quantified in brain by generation of tdTomato fluorescent protein signal from Ai9 reporter mice |
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2023-05-10
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In Vivo
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Comparing CRISPR/Cas9 gene editing efficencies between AAV9 and AAVcc47 in Ai9 mice with a 1:1 Cas9 to sgRNA ratio (CMV promoter) and self complementary sgRNA vector.
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A dual vector strategy was employed: one self complementary vector delivering two single guide RNAs targeting the Rosa26 locus and one delivering CMV driven SaCas9 (single stranded vector). This strategy was evaluted with both AAV9 (n=4) and AAVcc47 (n=4) by intravenous injection in Ai9 mice. A total dose of 4e12vg was injected into each mouse and vectors mixed in a 1:1 ratio of cas9 to guide RNA (2e12vg of CMV Sacas9 vector and 2e12vg of the self complementary sgRNA vector) and organs were harvested 4 weeks post injection. Editing efficency was determined by calculating percent TdTomato+ cells normalized to Dapi+ cells in liver and heart. |
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2021-09-16
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In Vitro
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Evaluation of DNA damage, cellular toxicity and inflammatory markers following saCas9 and gRNAs delivery by AAV2 in kidney organoids.
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AAV2 that carries saCas9 and AAV2 carrying two gRNAs against DMD gene and mCherry were used in kidney organoids to assess toxicity compared to AAV2-GFP or mock transduced kidney organoids. |
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2023-09-22
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In Vivo
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FUS (focused ultrasound) array validation in Ai9 mice
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9.3 week-old Ai9 mice (4 male and 4 female) were administered Ai9-targeting SaCas9 AAV9 vector through intravenous adminsitration (2E12 vg/mouse) and left hemisphere was targeted by FUS (focused ultrasound) array for BBB (blood brain barrier) opening |
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2022-04-15
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In Vivo
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Comparing CRISPR/Cas9 gene editing efficencies between AAV9 and AAVcc47 in Ai9 mice with a 1:1 Cas9 to sgRNA ratio (CB promoter)
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A dual vector strategy was employed: one delivering a single guide RNA and CB driven SaCas9, and another delivering the second guide RNA and CB driven SaCas9. This strategy was evaluted with both AAV9 (n=4) and AAVcc47 (n=5) by intravenous injection in Ai9 mice. A total dose of 2e12vg was injected into each mouse (1e12vg each vector mixed 1:1) and organs were harvested 4 weeks post injection. Editing efficency was determined by calculating percent TdTomato+ cells normalized to Dapi+ cells in liver and heart. |
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2021-09-16
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In Vivo
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Comparing CRISPR/Cas9 gene editing efficiencies between AAV9 and AAVcc47 in Ai9 mice with a 1:3 Cas9 to sgRNA ratio (CMV promoter)
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A dual vector strategy was employed: one delivering two single guide RNAs targeting the Rosa26 locus and one delivering CMV driven SaCas9 (both single stranded AAV cassettes). This strategy was evaluted with both AAV9 (n=4) and AAVcc47 (n=5) by intravenous injection in Ai9 mice. A total dose of 4e12vg was injected into each mouse and vectors mixed in a 1:4 ratio of cas9 to guide RNA (1e12vg of CMV Sacas9 vector and 3e12vg of the sgRNA vector) and organs were harvested 4 weeks post injection. Editing efficency was determined by calculating percent TdTomato+ cells normalized to Dapi+ cells in liver and heart. |
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2021-09-16
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In Vivo
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Testing virus region 8 (VR8) mutant cross-species compatible Adeno Associated Viruses (ccAAVs) in mice.
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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 a GFP reporter. The biodistribution of of virus transduction was chacterized in various tissues and cell types by fluorescence imaging quantification. |
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2020-11-19
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In Vivo
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Testing AAV5 for activation of tdTomato in mouse airway club and ciliated cells
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AAV2/5 mediated gene editing in the mouse airway was tested by deliverying SpCas9 and guide RNAs targeting the Ai9 transgene in Ai9 transgenic mice. Gene editing quantified by tdTomato activation and cell specific markers for club and ciliated cell types. |
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2021-09-21
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In Vitro
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Selection of gRNA sequences and gRNA scaffold modification lead to improved editing of the Ai9 locus in vitro
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Reporter transgene activation by SaCas9 gRNA target and modified scaffold sequences by transient transfection in immortalized Ai9 mouse fibroblasts |
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2021-04-17
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In Vivo
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[Validation]
Independent validation for Gao Delivery Team: Testing ssAAV5 delivered intratracheally for editing activity in lung epithelia in Ai9 mice
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AAV5 encoding CRISPR/Cas editing machinery were delivered to the lungs of reporter mice by intratracheal instillation. After 4 weeks incubation, the mice were dissected and the lungs imaged for the presence of tdTomato fluorescence, indicating successful editing. Editing calculated by dividing the number of tdTomato+ red cells by the number of nuclei in each airway |
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2021-03-30
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