Recombinant adeno-associated virus (rAAV) vectors are widely used for gene therapeutics, yet their early effects on host chromatin remodeling and nuclear organization remain insufficiently characterized. In contrast, several DNA viruses are known to remodel host chromatin accessibility, for example, Herpes simplex virus type 1 (HSV-1) in mammalian cells and baculoviruses in insect systems. Here, we evaluated genome accessibility and nuclear organization of primary human fibroblast cells at 24 and 48 h after infection with wild-type AAV2, single-stranded (ss) and self-complementary rAAV2, as well as ssAAV2 and ssAAV-DJ in human lung carcinoma cells. Genome-wide assay for transposase-accessible chromatin using sequencing showed no detectable change in host chromatin accessibility at either time point. A DNase I digestion assay at five candidate loci supported this observation. Although host accessibility remained stable, immunofluorescence-based single-cell analyses uncovered modest changes in histone abundance, RNA polymerase II–associated signals, nuclear morphology, and the organization of liquid–liquid phase-separated nuclear compartments. These effects were generally mild in primary fibroblasts but showed greater dependence on vector type, cell cycle state, and transgene expression in carcinoma cells. Collectively, at the tested doses and times, AAV-based vectors did not remodel chromatin accessibility at scale and induced only small changes in polymerase-associated readouts and nuclear architecture. These data are consistent with limited nuclear perturbation under these conditions.
BurdettT, NuseibehS. Changing trends in the development of AAV-based gene therapies: A meta-analysis of past and present therapies. Gene Ther2023;30(3–4):323–335; doi: 10.1038/s41434-022-00363-0
3.
PipeSW, LeebeekFWG, RechtM, et al.Gene therapy with etranacogene dezaparvovec for hemophilia B. N Engl J Med2023;388(8):706–718; doi: 10.1056/NEJMoa2211644
4.
Zwi-DantsisL, MohamedS, MassaroG, et al.Adeno-associated virus vectors: Principles, practices, and prospects in gene therapy. Viruses2025;17(2):239; doi: 10.3390/v17020239
5.
SamulskiRJ, MuzyczkaN. AAV-mediated gene therapy for research and therapeutic purposes. Annu Rev Virol2014;1(1):427–451; doi: 10.1146/annurev-virology-031413-085355
6.
Gil-FarinaI, FronzaR, KaeppelC, et al.Recombinant AAV integration is not associated with hepatic genotoxicity in nonhuman primates and patients. Mol Ther2016;24(6):1100–1105; doi: 10.1038/mt.2016.52
7.
KaeppelC, BeattieSG, FronzaR, et al.A largely random AAV integration profile after LPLD gene therapy. Nat Med2013;19(7):889–891; doi: 10.1038/nm.3230
8.
McCartyDM, YoungSM, SamulskiRJ. Integration of Adeno-Associated Virus (AAV) and recombinant AAV vectors. Annu Rev Genet2004;38(1):819–845; doi: 10.1146/annurev.genet.37.110801.143717
9.
NakaiH, YantSR, StormTA, et al.Extrachromosomal recombinant adeno-associated virus vector genomes are primarily responsible for stable liver transduction in vivo. J Virol2001;75(15):6969–6976; doi: 10.1128/JVI.75.15.6969-6976.2001
10.
SchneppBC, JensenRL, ChenCL, et al.Characterization of adeno-associated virus genomes isolated from human tissues. J Virol2005;79(23):14793–14803; doi: 10.1128/JVI.79.23.14793-14803.2005
11.
Penaud-BudlooM, Le GuinerC, NowrouziA, et al.Adeno-associated virus vector genomes persist as episomal chromatin in primate muscle. J Virol2008;82(16):7875–7885; doi: 10.1128/JVI.00649-08
12.
WesthausA, Cabanes-CreusM, RybickiA, et al.High-throughput in vitro, ex vivo, and in vivo screen of adeno-associated virus vectors based on physical and functional transduction. Hum Gene Ther2020;31(9–10):575–589; doi: 10.1089/hum.2019.264
13.
SchneppBC, ChulayJD, YeGJ, et al.Recombinant adeno-associated virus vector genomes take the form of long-lived, transcriptionally competent episomes in human muscle. Hum Gene Ther2016;27(1):32–42; doi: 10.1089/hum.2015.136
14.
KongX, WeiG, ChenN, et al.Dynamic chromatin accessibility profiling reveals changes in host genome organization in response to baculovirus infection. PLoS Pathog2020;16(6):e1008633; doi: 10.1371/journal.ppat.1008633
15.
CalléA, UgrinovaI, EpsteinAL, et al.Nucleolin is required for an efficient herpes simplex virus type 1 infection. J Virol2008;82(10):4762–4773; doi: 10.1128/JVI.00077-08
16.
GuH, RoizmanB. The degradation of promyelocytic leukemia and Sp100 proteins by herpes simplex virus 1 is mediated by the ubiquitin-conjugating enzyme UbcH5a. Proc Natl Acad Sci U S A2003;100(15):8963–8968; doi: 10.1073/pnas.1533420100
17.
XiaoX, LiJ, SamulskiRJ. Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus. J Virol1998;72(3):2224–2232; doi: 10.1128/JVI.72.3.2224-2232.1998
18.
SutterSO, MarconiP, MeierAF. Herpes Simplex Virus Growth, Preparation, and Assay. In: Herpes Simplex Virus : Methods and Protocols. (DiefenbachRJ, FraefelC., eds.) Springer: New York, NY; 2020, pp. 57–72. (Methods in Molecular Biology). 10.1007/978-1-4939-9814-2_3
19.
WingettSW, AndrewsS. FastQ Screen: A tool for multi-genome mapping and quality control. F1000Res2018;7:1338; doi: 10.12688/f1000research.15931.2
LangmeadB, SalzbergSL. Fast gapped-read alignment with Bowtie 2. Nat Methods2012;9(4):357–359; doi: 10.1038/nmeth.1923
22.
HatakeyamaM, OpitzL, RussoG, et al.SUSHI: An exquisite recipe for fully documented, reproducible and reusable NGS data analysis. BMC Bioinformatics2016;17(1):228; doi: 10.1186/s12859-016-1104-8
23.
BolgerAM, LohseM, UsadelB. Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics2014;30(15):2114–2120; doi: 10.1093/bioinformatics/btu170
24.
AmemiyaHM, KundajeA, BoyleAP. The ENCODE blacklist: Identification of problematic regions of the genome. Sci Rep2019;9(1):9354; doi: 10.1038/s41598-019-45839-z
25.
RamírezF, RyanDP, GrüningB, et al.deepTools2: A next generation web server for deep-sequencing data analysis. Nucleic Acids Res2016;44(W1):W160–W165; doi: 10.1093/nar/gkw257
26.
LiH, HandsakerB, WysokerA, 1000 Genome Project Data Processing Subgroup. et al.; The sequence alignment/map format and SAMtools. Bioinformatics2009;25(16):2078–2079; doi: 10.1093/bioinformatics/btp352
27.
ZhangY, LiuT, MeyerCA, et al.Model-based analysis of ChIP-Seq (MACS). Genome Biol2008;9(9):R137; doi: 10.1186/gb-2008-9-9-r137
28.
EwelsP, MagnussonM, LundinS, et al.MultiQC: Summarize analysis results for multiple tools and samples in a single report. Bioinformatics2016;32(19):3047–3048; doi: 10.1093/bioinformatics/btw354
WickhamH. ggplot2: Elegant Graphics for Data Analysis, 2nd ed. Springer International Publishing: Cham; 2016. Imprint: Springer; 2016. 1 p. (Use R!); doi: 10.1007/978-3-319-24277-4
31.
WickhamH, AverickM, BryanJ, et al.Welcome to the tidyverse. JOSS2019;4(43):1686; doi: 10.21105/joss.01686
32.
SofroniewN, LambertT, EvansK, et al.napari: A multi-dimensional image viewer for Python. Zenodo; 2022; doi:10.5281/ZENODO.7276432. Available from: https://zenodo.org/record/7276432
33.
SchindelinJ, Arganda-CarrerasI, FriseE, et al.Fiji: An open-source platform for biological-image analysis. Nat Methods2012;9(7):676–682; doi: 10.1038/nmeth.2019
34.
RämöP, SacherR, SnijderB, et al.CellClassifier: Supervised learning of cellular phenotypes. Bioinformatics2009;25(22):3028–3030; doi: 10.1093/bioinformatics/btp524
Posit team. RStudio: Integrated Development Environment for R. Posit Software, PBC: Boston, MA; 2025. Available from: http://www.posit.co/.
37.
Nepon-SixtB, AlexandrowM. DNase I chromatin accessibility analysis. Bio Protoc2019;9(23):e3444; doi: 10.21769/BioProtoc.3444
38.
KremerJR, MastronardeDN, McIntoshJR. Computer visualization of three-dimensional image data using IMOD. J Struct Biol1996;116(1):71–76; doi: 10.1006/jsbi.1996.0013
39.
PunjaniA, RubinsteinJL, FleetDJ, et al.cryoSPARC: Algorithms for rapid unsupervised cryo-EM structure determination. Nat Methods2017;14(3):290–296; doi: 10.1038/nmeth.4169
40.
FetniR, RicherCL, MalfoyB, et al.Cytologic characterization of two distinct alpha satellite DNA domains on human chromosome 7, using double-labeling hybridizations in fluorescence and electron microscopy on a melanoma cell line. Cancer Genet Cytogenet1997;96(1):17–22; doi: 10.1016/S0165-4608(96)00282-8
41.
HennigT, MichalskiM, RutkowskiAJ, et al.HSV-1-induced disruption of transcription termination resembles a cellular stress response but selectively increases chromatin accessibility downstream of genes. PLoS Pathog2018;14(3):e1006954; doi: 10.1371/journal.ppat.1006954
42.
NarotamoH, FernandesMS, MoreiraAM, et al.A machine learning approach for single cell interphase cell cycle staging. Sci Rep2021;11(1):19278; doi: 10.1038/s41598-021-98489-5
43.
StephensAD, LiuPZ, BaniganEJ, et al.Chromatin histone modifications and rigidity affect nuclear morphology independent of lamins. Mol Biol Cell2018;29(2):220–233; doi: 10.1091/mbc.E17-06-0410
44.
HindererC, KatzN, BuzaEL, et al.Severe toxicity in nonhuman primates and piglets following high-dose intravenous administration of an adeno-associated virus vector expressing human SMN. Hum Gene Ther2018;29(3):285–298; doi: 10.1089/hum.2018.015
SchwartzRA, CarsonCT, SchuberthC, et al.Adeno-associated virus replication induces a DNA damage response coordinated by DNA-dependent protein kinase. J Virol2009;83(12):6269–6278; doi: 10.1128/JVI.00318-09
Gonzalez-SandovalA, PekrunK, TsujiS, et al.The AAV capsid can influence the epigenetic marking of rAAV delivered episomal genomes in a species dependent manner. Nat Commun2023;14(1):2448; doi: 10.1038/s41467-023-38106-3
49.
KianiK, SanfordEM, GoyalY, et al.Changes in chromatin accessibility are not concordant with transcriptional changes for single‐factor perturbations. Mol Syst Biol2022;18(9):e10979; doi: 10.15252/msb.202210979
50.
YamazakiT, LiuL, ManleyJL. Oxidative stress induces Ser 2 dephosphorylation of the RNA polymerase II CTD and premature transcription termination. Transcription2021;12(5):277–293; doi: 10.1080/21541264.2021.2009421
51.
GulyasL, GlaunsingerBA. RNA polymerase II subunit modulation during viral infection and cellular stress. Curr Opin Virol2022;56:101259; doi: 10.1016/j.coviro.2022.101259
52.
FranzosoFD, SeyffertM, VogelR, et al.Cell cycle-dependent expression of Adeno-Associated Virus 2 (AAV2) rep in coinfections with Herpes Simplex Virus 1 (HSV-1) gives rise to a mosaic of cells replicating either AAV2 or HSV-1. J Virol2017;91(15):e00357–e00317; doi: 10.1128/JVI.00357-17
53.
ZubkovaES, BeloglazovaIB, RatnerEI, et al.Transduction of rat and human adipose-tissue derived mesenchymal stromal cells by adeno-associated viral vector serotype DJ. Biol Open2021;10(9):bio058461; doi: 10.1242/bio.058461
54.
BatieM, FrostJ, FrostM, et al.Hypoxia induces rapid changes to histone methylation and reprograms chromatin. Science2019;363(6432):1222–1226; doi: 10.1126/science.aau5870
55.
RytkönenKT, FauxT, MahmoudianM, et al.Histone H3K4me3 breadth in hypoxia reveals endometrial core functions and stress adaptation linked to endometriosis. iScience2022;25(5):104235; doi: 10.1016/j.isci.2022.104235
56.
BoulonS, WestmanBJ, HuttenS, et al.The Nucleolus under Stress. Mol Cell2010;40(2):216–227; doi: 10.1016/j.molcel.2010.09.024
57.
SutterSO, LkharraziA, SchranerEM, et al.Adeno-associated virus type 2 (AAV2) uncoating is a stepwise process and is linked to structural reorganization of the nucleolus. PLoS Pathog2022;18(7):e1010187; doi: 10.1371/journal.ppat.1010187
58.
SahinU, FerhiO, JeanneM, et al.Oxidative stress–induced assembly of PML nuclear bodies controls sumoylation of partner proteins. J Cell Biol2014;204(6):931–945; doi: 10.1083/jcb.201305148
59.
SchererM, ReadC, NeusserG, et al.Dual signaling via interferon and DNA damage response elicits entrapment by giant PML nuclear bodies. Elife2022;11:e73006; doi: 10.7554/eLife.73006
60.
GalganskiL, UrbanekMO, KrzyzosiakWJ. Nuclear speckles: Molecular organization, biological function and role in disease. Nucleic Acids Res2017;45(18):10350–10368; doi: 10.1093/nar/gkx759
61.
SungH-M, SchottJ, BossP, et al.Stress-induced nuclear speckle reorganization is linked to activation of immediate early gene splicing. J Cell Biol2023;222(12):e202111151; doi: 10.1083/jcb.202111151
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