This readme file was generated on 2025-02-18 ------------------- GENERAL INFORMATION ------------------- Title of Dataset: Data from: Adenine base editing rescues pathogenic phenotypes in tissue engineered vascular model of Hutchinson-Gilford Progeria Syndrome Author Contact Information (Name, Institution, Email, ORCID) Principal Investigator: George A. Truskey Institution: Duke University, Department of Biomedical Engineering Email: gtruskey@duke.edu ORCID: 0000-0002-6885-4489 Associate or Co-investigator: Nadia O. Abutaleb Institution: Duke University, Department of Biomedical Engineering Email: nadia@abutaleb.com ORCID: 0000-0002-0995-5730 Associate or Co-investigator: Xin D. Gao Institution: Broad Institute of Massachusetts Institute of Technology and Harvard University Email: xgao@broadinstitute.org ORCID: 0000-0003-2917-2060 Associate or Co-investigator: Akhil Bedapudi Institution: Duke University, Department of Biomedical Engineering Email: akhilbedapudi@gmail.com ORCID: 0000-0001-8008-5822 Associate or Co-investigator: Leandro Choi Institution: Emory University Email: leandro.choi@emory.edu ORCID: 0000-0001-8965-9896 Associate or Co-investigator: Kevin L. Shores Institution: Duke University, Department of Biomedical Engineering Email: kevin.shores@duke.edu ORCID: 0000-0002-6205-8318 Associate or Co-investigator: Crystal Kennedy Institution: Duke University, University Program in Genetics and Genomics Email: crystal.kennedy@duke.edu ORCID: 0000-0003-3405-3381 Associate or Co-investigator: Jordyn E. Duby Institution: Harvard University Email: jduby@college.harvard.edu ORCID: 0009-0001-2638-2946 Associate or Co-investigator: Kan Cao Institution: University of Maryland at College Park, Department of Cell Biology and Molecular Genetics Email: kcao@umd.edu Associate or Co-investigator: David R. Liu Institution: Broad Institute of Massachusetts Institute of Technology and Harvard University Email: drliu@fas.harvard.edu * Date of data collection (single date, range, approximate date): 2021-09-01 to 2923-12-01 * Geographic location of data collection (if applicable): FCIEMAS rooms 1305 and 1313, Duke University. Sequencing: David Liu lab, Broad Institute, Cambridge, MA * Funding and grant numbers (if applicable): NIH Grant R01 HL138252-01 (GAT), NIH Grant NIH Grant UH3TR002142 (GAT), and NSF GRFP grant DGE1644868 (NOA). Next Generation Sequencing - FASTQ files are available from the National Center for Biotechnology Information's Sequence Read Archive under BioProject (PRJNA1148490) at https://ncbi.nlm.nih.gov/bioproject/?term=PRJNA1148490. This dataset includes data from experiments to support a publication in APL Bioengineering, doi: 10.1063/5.0244026. -------------------- DATA & FILE OVERVIEW -------------------- File list (filenames, directory structure (for zipped files) and brief description of all data files): 1. Fig 1B-E iPSC-ABE7.10max_DNA sequencing – Results of high throughput DNA sequencing on iPSCs treated with ABE7.10max including percent wildtype (% G at protospacer position 6), % G at protospacer position 10 and insertion and deletions (indels). - Appears as Figure 1, panels B, C, D, and E. - Next Generation Sequencing performed by Daniel Gao High throughput DNA sequencing - FASTQ files are available from the National Center for Biotechnology Information's Sequence Read Archive under BioProject (PRJNA1148490). - Data analyzed by Nadia Abutaleb and Daniel Gao - Reported in Nadia Abutaleb’s dissertation – Tissue Engineered Blood Vessels to Study Endothelial Dysfunction in Hutchinson-Gilford Progeria Syndrome, April 2022; Duke University ProQuest Dissertations & Theses, 2022. 29060759 2. ANOVA_Fig 1B-D – Statistical analysis of data sets in item 1, Figs 1 B-D. 3. FigS3 – DNA sequencing of viECs viSMCs - DNA sequencing on 168 CL2 and HGPS 003 CL1D viECs (p1) and viSMCs (p4) - Appears as Figure S3, panels A-F. - Data collected by Nadia Abutaleb - Next Generation Sequencing performed by Daniel Gao - FASTQ files are available from the National Center of Biotechnology’s Information Sequence Read Archive under BioProject (PRJNA1148490). - Reported in Nadia Abutaleb’s dissertation – Tissue Engineered Blood Vessels to Study Endothelial Dysfunction in Hutchinson-Gilford Progeria Syndrome, April 2022; Duke University ProQuest Dissertations & Theses, 2022. 29060759 4. ANOVA_FigS3 - Statistical analysis of data sets in item 3. 5. Fig2AE – Nuclear blebbing of healthy viECs, HGPS viECs, and edited HGPS viECs. - Appears as Figure 2, panels A, E. - Data collected by Nadia Abutaleb - Reported in Nadia Abutaleb’s dissertation – Tissue Engineered Blood Vessels to Study Endothelial Dysfunction in Hutchinson-Gilford Progeria Syndrome, April 2022; Duke University ProQuest Dissertations & Theses, 2022. 29060759 6. Fig 2BF DCFDA – measurement of reactive oxygen species (ROS) healthy, HGPS viECs, and edited HGPS viECs - Appears as Figure 2, panels B, F. - Data collected by Nadia Abutaleb Reported in Nadia Abutaleb’s dissertation – Tissue Engineered Blood Vessels to Study Endothelial Dysfunction in Hutchinson-Gilford Progeria Syndrome, April 2022; Duke University ProQuest Dissertations & Theses, 2022. 29060759 7. Fig 2CDGH and Fig S4AKi67and gH2ax – Measures of proliferation (Ki67) and double stranded breaks (gH2ax) of healthy, HGPS viECs, and edited HGPS viECs. - Appears as Figure 2, panels C, G (Ki67); D, H (gH2ax positive cells); Figure S4A, C (gH2ax foci per cell); S4B, D (gH2ax foci per positive nuclei). - Data collected by Nadia Abutaleb - Reported in Nadia Abutaleb’s dissertation – Tissue Engineered Blood Vessels to Study Endothelial Dysfunction in Hutchinson-Gilford Progeria Syndrome, April, 2022; Duke University ProQuest Dissertations & Theses, 2022. 29060759. 8. ANOVA_Fig2 and ANOVA_FigS4 - Statistical analysis of data sets in items 5, 6, and 7. 9. Fig3 - Effect of fluid shear stress on nitric oxide production (DAF-FM) and gene expression of healthy, HGPS viECs, and edited HGPS viECs under static conditions or exposed to 12 dynes/cm2 shear stress for 24 hours. - Appears as Figure 3, panels A (DAF-FM), or endothelial genes (B) NOS3, (C) KLF2, (D) NRF2, (E) NQO1, (F) TXNRD1, (G) GCLM and (H) GCLC. - Data collected by Nadia Abutaleb - Reported in Nadia Abutaleb’s dissertation – Tissue Engineered Blood Vessels to Study Endothelial Dysfunction in Hutchinson-Gilford Progeria Syndrome, April 2022; Duke University ProQuest Dissertations & Theses, 2022. 29060759. 10. ANOVA_Fig3 - Statistical analysis of data sets in item 9. 11. Fig 4 - Effect of ABE7.10max treatment on vasoactivity of TEBVs made with edited HGPS viECs and viSMCs. - Appears as Figure 4 panels A (1 week), B (2 weeks) and C (3 weeks) - Data collected by Nadia Abutaleb - Reported in Nadia Abutaleb’s dissertation – Tissue Engineered Blood Vessels to Study Endothelial Dysfunction in Hutchinson-Gilford Progeria Syndrome, April 2022; Duke University ProQuest Dissertations & Theses, 2022. 29060759. 12. ANOVA_Fig4 - Statistical analysis of data sets in item 11. 13. Fig 5DEF - Effect of ABE7.10max treatment on α-smooth muscle actin (αSMA) smooth muscle cell myosin heavy chain 11 (MHC11), and endothelial cell vWF in TEBVs fabricated with healthy cells, HGPS cells, or HGPS cells treated with ABE7.10max + LMNA mutation-targeting guide RNA (sgPro). - Appears as Figure 5 panels D (αSMA), E (MHC11) and F (vWF) - Data collected by Nadia Abutaleb - Reported in Nadia Abutaleb’s dissertation – Tissue Engineered Blood Vessels to Study Endothelial Dysfunction in Hutchinson-Gilford Progeria Syndrome, April 2022; Duke University ProQuest Dissertations & Theses, 2022. 29060759. 14. ANOVA_Fig5DEF - Statistical analysis of data sets in item 14. 15. Fig 6CD Effect of ABE7.10max treatment on fibronectin (FN) and collagen IV expression in TEBVs fabricated with healthy cells, HGPS cells, or HGPS cells treated with ABE7.10max + LMNA mutation-targeting guide RNA (sgPro). - Appears as Figure 6 panels C (FN) and D (Collagen IV) - Data collected by Nadia Abutaleb - Reported in Nadia Abutaleb’s dissertation – Tissue Engineered Blood Vessels to Study Endothelial Dysfunction in Hutchinson-Gilford Progeria Syndrome, April 2022; Duke University ProQuest Dissertations & Theses, 2022. 29060759. 16. ANOVA_Fig6 - Statistical analysis of data sets in item 17. 17. Fig 7 - Fraction of edited and unedited viECs and viSMCs - Appears as Figure 7 panels A (viSMCs) and B (viECs) - Data collected by Nadia Abutaleb - Reported in Nadia Abutaleb’s dissertation – Tissue Engineered Blood Vessels to Study Endothelial Dysfunction in Hutchinson-Gilford Progeria Syndrome, April 2022; Duke University ProQuest Dissertations & Theses, 2022. 29060759. 18. ANOVA_Fig7 - Statistical analysis of data sets in item 17. 19. Fig 8- Effect of ratio of edited: unedited HGPS cells on TEBVs after 3 and 5 weeks of perfusion. - Appears as Figure 8 panels A (vasoconstriction), B (vasodilation), C (viSMC density), D (Ki 67), E (αSMA), F (MHC11), G (Fraction Wild Type Allele) - Data collected by Akhil Bedapudi - Data analyzed by Akhil Bedapudi, Kevin Shores and George Truskey 20. ANOVA_Fig8 - Statistical analysis of data sets in item 19. 21. Fig 8G sequences - Sanger sequencing of TEBVs made with different ratios of edited and unedited cells. - Experiments performed by Akhil Bedapudi - Sanger sequencing performed by Azenta/GeneWiz 7/2023. - Data analyzed by George Truskey - Excel file Figure 8G Samples.xlsx listing samples and experimental conditions. - Sequence files ending in .sequence and ab1 can be read using SnapGene Viewer which can be downloaded from www.snapgene.com without charge 22. Fig S5 - Effect of ABE7.10max treatment on viSMC replication and cell density. - Appears as Figure S5 panels C (nuclei/mm2) and D (Ki67+ cells/mm2) - Data collected by Nadia Abutaleb - Reported in Nadia Abutaleb’s dissertation – Tissue Engineered Blood Vessels to Study Endothelial Dysfunction in Hutchinson-Gilford Progeria Syndrome, April 2022; Duke University ProQuest Dissertations & Theses, 2022. 29060759. 23. ANOVA_FigS5 - Statistical analysis of data sets in item 22. 24. Fig S6 - Off-target editing in viECs and viSMCs containing different ratios of edited and unedited cells - Appears as Figure S6 (A, B) Bystander editing of G at position 10 and (C, D) percent Indels over time for viECs (A, C) and viSMCs (B, D) in co-cultures of edited sgPro and unedited HGPS cells 25. ANOVA_FigS6 Statistical analysis of data sets in item 24. -------------------------- METHODOLOGICAL INFORMATION -------------------------- The rare, accelerated aging disease Hutchinson-Gilford Progeria Syndrome (HGPS) is commonly caused by a de novo c.1824 C>T point mutation of the LMNA gene that results in the protein progerin. The primary cause of death is a heart attack or stroke arising from atherosclerosis. A characteristic feature of HGPS arteries is loss of smooth muscle cells. The goal of this project was to assess whether base editing correction of human HGPS tissue engineered blood vessels (TEBVs) prevents the HGPS vascular phenotype and to identify the minimum fraction of edited smooth muscle cells needed to effect such changes, we transduced HGPS iPSCs with lentivirus containing ABE7.10max base editor. Endothelial cells (viECs) and smooth muscle cells (viSMCs) obtained by differentiation of iPSCs did not express progerin, and had double-stranded DNA breaks and reactive oxygen species to levels in healthy viSMCs and viECs. Editing restored a normal response to shear stress in edited viECs. Normal vasodilation and viSMC density were restored in TEBVs made with edited cells. When TEBVs were prepared with at least 50% edited smooth muscle cells, viSMC proliferation and myosin heavy chain levels significantly improved. Sequencing of TEBV cells after perfusion indicated an enrichment of edited cells after 5 weeks of perfusion when they comprised 50% of the initial number of cells in the TEBVs. Thus, base editing correction of a fraction of HGPS vascular cells improves human TEBV phenotype. Statistical analysis was performed using JMP Pro 14 (SAS). Data were analyzed using a one- or two-way ANOVA and post-hoc Tukey tests for pairwise comparisons. For time-dependent assays on the same sample, repeated measures ANOVA was used. Results were plotted using GraphPad Prism. Data are represented as mean ± SD with N = number of TEBVs or number of independent experiments for 2D cell culture experiments; p ≤ 0.05 was considered significant. ------------------------- STANDARDS AND CALIBRATION INFORMATION -------------------------- Antibodies were validated by the manufacturer. The smooth muscle and endothelial cells derived from patients with progeria served as controls for the expression of progerin and the presence of the c.1824 C>T point mutation of the LMNA gene. Missing data treatments (null, -99, na, etc.): null ------------------------- USE and ACCESS INFORMATION -------------------------- Data License: CC0 Other Rights Information: To cite the data: Abutaleb, N. O., Gao, X. D., Bedapudi, A., Choi, L., Shores, K. L., Kennedy, C., Duby, J. E., Cao, K., Liu, D. R., & Truskey, G. A. (2025). Data from: Adenine base editing rescues pathogenic phenotypes in tissue engineered vascular model of Hutchinson-Gilford Progeria Syndrome. Duke Research Data Repository. https://doi.org/10.7924/r4pg1xv1b