Evaluation of DNA damage, cellular toxicity and inflammatory markers following saCas9 and gRNAs delivery by AAV2 in kidney organoids.

Experiment: Evaluation of DNA damage, cellular toxicity and inflammatory markers following saCas9 and gRNAs delivery by AAV2 in kidney organoids.

PI: Ryuji Morizane, MD, PhD

Description: 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.

Biomarker Assay:

KIM-1, MCP-1, NGAL, IL1-beta, IL-6, and IL10 levels were measured using the Meso Scale Discovery (MSD) platform
Toxicity was caclulated as the percent of KIM-1 (HAVCR1) positivity in LTL+ cells
Compartmental DNA damage was calculated as the percent of YH2AX co-staining among LTL, CHD1 and MEIS1/2/3 positive cells

Parent Project: Vascularized kidney organoids on chip for efficacy and toxicity testing of somatic genome editing

Other experiments in this project: 6

Download: Submitted files

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Editors

SaCas9 (AAV-SaCas9)

Guide Target Locus

DMD

Guides

DMD low gRNA-2

Delivery Systems

AAV

Models

Human kidney organoid (stem cell-derived)

Vectors

AAV2-gRNA

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Results

Evaluation_of_DNA_damage,_cellular_toxicity_and_inflammatory_markers_following_saCas9_and_gRNAs_delivery_by_AAV2_in_kidney_organoids.
								Other Measurement
Record Id	Condition	Editor	Model	Delivery	Target Locus	Guide	Vector	Dosage	Biomarker Detection %YH2AX+/LTL+ cells	Biomarker Detection %YH2AX+/CHD1+ cells	Biomarker Detection %YH2AX+/MEIS1/2/3+ cells	Biomarker Detection %KIM1+/LTL+ cells	Biomarker Detection KIM-1 (pg/mL)	Biomarker Detection MCP-1 (pg/mL)	Biomarker Detection NGAL (pg/mL)	Biomarker Detection IL1-beta (pg/mL)	Biomarker Detection IL-6 (pg/mL)	Biomarker Detection IL-10 (pg/mL)	Image
15000003441	no AAV control		Human kidney organoid (stem cell-derived)					MOI=10^5	0.218818380743982; 1.07962213225371; 0.845070422535211;	0.78125; 1.02040816326531; 0;	4.28134556574923; 1.98757763975155; 2.71565495207668;	25; 16.66667; 0; 14.2857; 20; 16.666667; 12.5;	51.3995733584671; 43.6964290781139; 66.0926756135368; 50.1231942837828; 38.8285557898405; 46.6445392989742; 46.4596779579597; 46.6990608708682;	5.0763176199031; 6.09109175920988; 10.7828875743226; 7.80897357375238; 5.31558434785989; 6.13127470317533; 8.15865512382412; 5.08765217318633;	1945.87177714803; 2378.00592659228; 2450.71409648479; 2402.33372573432; 1891.05748937221; 2587.61534903933; 2628.15126475537; 2431.52943287365;	0.989206294342492; 1.37804389357951; 2.63693681324954; 2.4051573924998; 2.20678733880701; 1.16206361649702; 2.68628458619969; 1.7561532152817;	1.65477687183361; 1.71337051363705; 3.94681048062236; 2.42342203553629; 2.45673546508562; 3.09580748201716; 3.14160853809432; 2.16536385889831;	446.542860760008; 455.804285816509; 482.350513357372; 428.17839303964; 468.837814509775; 457.181289899693; 482.46822882869; 425.77992425901;
15000003442	AAV2/2-GFP		Human kidney organoid (stem cell-derived)	AAV				MOI=10^5	64.3356643356643; 57.5418994413408; 52.6315789473684;	15.7534246575342; 29.8013245033113; 26.5625;	23.3995584988962; 32.9918032786885; 35.4285714285714;	50; 66.66667; 60; 80; 71.43; 62.5; 50;	71.5833165541194; 90.2160638029641; 59.3236870646692; 81.469906438934; 78.8138728778462; 63.8860238886115; 61.428165238845; 69.2591122635337;	18.0070916190554; 16.1686378082723; 8.70221005688366; 17.4432793219612; 32.4877578217399; 9.74665350939028; 16.4090371835311; 9.0209742116227;	3461.44354915398; 4007.01594029684; 2383.42759214056; 2606.11627788187; 3466.97334025547; 2910.32739554959; 4761.98444276883; 2834.6371167695;	4.33968406632017; 4.80608939258317; 3.74139531934747; 3.98690556903495; 4.89736567174163; 4.55175310293446; 3.77368074815052; 4.6430370516758;	7.72452780216676; 8.8784773684702; 5.51973586207241; 10.1474367515684; 7.97776945388018; 7.4266544119219; 7.43532003372835; 8.15770843055003;	512.276847412247; 490.682364581603; 513.103953627712; 450.859966622963; 515.578696906728; 479.309837807072; 511.275559120074; 457.67154745967;
15000003443	AAV2/2-SaCas9 + AAV2/2-gRNA	SaCas9 (AAV-SaCas9)	Human kidney organoid (stem cell-derived)	AAV	DMD	DMD low gRNA-2	AAV2-gRNA	MOI=10^5	87.1508379888268; 94.2028985507246; 86.6666666666667;	43.3333333333333; 77.4193548387097; 44.4444444444444;	29.375; 33.6538461538462; 41.5770609318996;	83.33333; 66.66667; 50; 75; 75; 75; 60;	73.0450520275434; 247.799688076254; 57.3500937297986; 51.5853862679727; 104.236737882331; 50.1516171305266; 102.742313385041; 126.625898599655;	10.552638994931; 10.0996567940629; 6.94973325618479; 6.54733914189619; 16.9785383180146; 6.82945998412296; 17.9601172079959; 9.57669773080734;	2240.35305387958; 3082.66840339688; 2182.40694910355; 2029.08363507414; 5895.0685121659; 2129.72429900216; 6258.05943091146; 5639.0439532151;	3.04035320059101; 3.45685924657181; 2.50921630343338; 2.51575347278385; 3.02762559397732; 2.63136714995803; 3.56041744685856; 3.69759523331727;	7.13986096462846; 7.51252056707063; 3.84216984250873; 5.13790801017772; 6.91414028649866; 5.20233895635352; 6.79682820460547; 7.06756386236927;	495.417375758954; 464.933192143006; 526.17876439772; 476.437321338346; 497.545747150637; 453.317100769354; 522.919777936497; 466.579803799361;

Toxicity and inflammatory responses to AAV2 and saCas9 genome editing. (C-F) Toxicity evaluation by immunohistochemistry. (G,H) Biomarker assays for toxicity and inflammation.

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Experiment: Evaluation of DNA damage, cellular toxicity and inflammatory markers following saCas9 and gRNAs delivery by AAV2 in kidney organoids.

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Results