Time-Restricted PiggyBac DNA Transposition by Transposase Protein Delivery Using Lentivirus-Derived Nanoparticles

Overview

Journal Mol Ther Nucleic Acids

Publisher Cell Press

Date 2018 Jun 3

PMID 29858060

Citations 9

Authors

Kristian Alsbjerg Skipper

Mathias Gaarde Nielsen

Sofie Andersen

Laura Barrett Ryo

Rasmus O Bak

Jacob Giehm Mikkelsen

Affiliations

Soon will be listed here.

Abstract

Continuous innovation of revolutionizing genome engineering technologies calls for an intensified focus on new delivery technologies that not only match the inventiveness of genome editors but also enable the combination of potent delivery and time-restricted action of genome-modifying bits and tools. We have previously demonstrated the use of lentivirus-derived nanoparticles (LNPs) as a protein delivery vehicle, incorporating and transferring DNA transposases, designer nucleases, or RNA-guided endonucleases fused to the N terminus of the Gag/GagPol polypeptide. Here, we establish LNP-directed transfer of the piggyBac DNA transposase protein by fusing the transposase to the integrase protein in the C-terminal end of GagPol. We show protein incorporation and proteolytic release of the DNA transposase within matured LNPs, resulting in high levels of DNA transposition activity in LNP-treated cells. Importantly, as opposed to conventional delivery methods based on transfection of plasmid DNA or in-vitro-transcribed mRNA, protein delivery by LNPs effectively results in time-restricted action of the protein (<24 hr) without compromising overall potency. Our findings refine LNP-directed piggyBac transposase delivery, at present the only available direct delivery strategy for this particular protein, and demonstrate a novel strategy for restricting and fine-tuning the exposure of the genome to DNA-modifying enzymes.

Citing Articles

Cell-targeted gene modification by delivery of CRISPR-Cas9 ribonucleoprotein complexes in pseudotyped lentivirus-derived nanoparticles.

Nielsen I, Rovsing A, Janns J, Thomsen E, Ruzo A, Boggild A Mol Ther Nucleic Acids. 2024; 35(4):102318.

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Engineered lentivirus-derived nanoparticles (LVNPs) for delivery of CRISPR/Cas ribonucleoprotein complexes supporting base editing, prime editing and in vivo gene modification.

Haldrup J, Andersen S, Labial A, Wolff J, Frandsen F, Skov T Nucleic Acids Res. 2023; 51(18):10059-10074.

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Delivering genes with human immunodeficiency virus-derived vehicles: still state-of-the-art after 25 years.

Wolff J, Mikkelsen J J Biomed Sci. 2022; 29(1):79.

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CRISPR-Cas9-directed gene tagging using a single integrase-defective lentiviral vector carrying a transposase-based Cas9 off switch.

Thomsen E, Skipper K, Andersen S, Haslund D, Skov T, Mikkelsen J Mol Ther Nucleic Acids. 2022; 29:563-576.

PMID: 36090759 PMC: 9403905. DOI: 10.1016/j.omtn.2022.08.005.

Integrase deficient lentiviral vector: prospects for safe clinical applications.

Yew C, Gurumoorthy N, Nordin F, Tye G, Wan Kamarul Zaman W, Tan J PeerJ. 2022; 10:e13704.

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References

Xie F, Ye L, Chang J, Beyer A, Wang J, Muench M . Seamless gene correction of β-thalassemia mutations in patient-specific iPSCs using CRISPR/Cas9 and piggyBac. Genome Res. 2014; 24(9):1526-33. PMC: 4158758. DOI: 10.1101/gr.173427.114. View

Ling C, Bhukhai K, Yin Z, Tan M, Yoder M, Leboulch P . High-Efficiency Transduction of Primary Human Hematopoietic Stem/Progenitor Cells by AAV6 Vectors: Strategies for Overcoming Donor-Variation and Implications in Genome Editing. Sci Rep. 2016; 6:35495. PMC: 5069717. DOI: 10.1038/srep35495. View

Wang J, Exline C, DeClercq J, Llewellyn G, Hayward S, Li P . Homology-driven genome editing in hematopoietic stem and progenitor cells using ZFN mRNA and AAV6 donors. Nat Biotechnol. 2015; 33(12):1256-1263. PMC: 4842001. DOI: 10.1038/nbt.3408. View

Cyranoski D . Chinese scientists to pioneer first human CRISPR trial. Nature. 2016; 535(7613):476-7. DOI: 10.1038/nature.2016.20302. View

Tebas P, Stein D, Tang W, Frank I, Wang S, Lee G . Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV. N Engl J Med. 2014; 370(10):901-10. PMC: 4084652. DOI: 10.1056/NEJMoa1300662. View

Kebriaei P, Singh H, Huls M, Figliola M, Bassett R, Olivares S . Phase I trials using Sleeping Beauty to generate CD19-specific CAR T cells. J Clin Invest. 2016; 126(9):3363-76. PMC: 5004935. DOI: 10.1172/JCI86721. View

Jakobsen M, Stenderup K, Rosada C, Moldt B, Kamp S, Dam T . Amelioration of psoriasis by anti-TNF-alpha RNAi in the xenograft transplantation model. Mol Ther. 2009; 17(10):1743-53. PMC: 2835022. DOI: 10.1038/mt.2009.141. View

Lee C, Li J, Liou J, Charng Y, Huang Y, Lee H . A gene delivery system for human cells mediated by both a cell-penetrating peptide and a piggyBac transposase. Biomaterials. 2011; 32(26):6264-76. DOI: 10.1016/j.biomaterials.2011.05.012. View

Wilber A, Wangensteen K, Chen Y, Zhuo L, Frandsen J, Bell J . Messenger RNA as a source of transposase for sleeping beauty transposon-mediated correction of hereditary tyrosinemia type I. Mol Ther. 2007; 15(7):1280-7. DOI: 10.1038/sj.mt.6300160. View

10.

Ran F, Cong L, Yan W, Scott D, Gootenberg J, Kriz A . In vivo genome editing using Staphylococcus aureus Cas9. Nature. 2015; 520(7546):186-91. PMC: 4393360. DOI: 10.1038/nature14299. View

11.

Woltjen K, Michael I, Mohseni P, Desai R, Mileikovsky M, Hamalainen R . piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature. 2009; 458(7239):766-70. PMC: 3758996. DOI: 10.1038/nature07863. View

12.

Yin H, Song C, Dorkin J, Zhu L, Li Y, Wu Q . Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo. Nat Biotechnol. 2016; 34(3):328-33. PMC: 5423356. DOI: 10.1038/nbt.3471. View

13.

Kaji K, Norrby K, Paca A, Mileikovsky M, Mohseni P, Woltjen K . Virus-free induction of pluripotency and subsequent excision of reprogramming factors. Nature. 2009; 458(7239):771-5. PMC: 2667910. DOI: 10.1038/nature07864. View

14.

Ye L, Wang J, Beyer A, Teque F, Cradick T, Qi Z . Seamless modification of wild-type induced pluripotent stem cells to the natural CCR5Δ32 mutation confers resistance to HIV infection. Proc Natl Acad Sci U S A. 2014; 111(26):9591-6. PMC: 4084478. DOI: 10.1073/pnas.1407473111. View

15.

Woltjen K, Hamalainen R, Kibschull M, Mileikovsky M, Nagy A . Transgene-free production of pluripotent stem cells using piggyBac transposons. Methods Mol Biol. 2011; 767:87-103. DOI: 10.1007/978-1-61779-201-4_7. View

16.

Ding S, Wu X, Li G, Han M, Zhuang Y, Xu T . Efficient transposition of the piggyBac (PB) transposon in mammalian cells and mice. Cell. 2005; 122(3):473-83. DOI: 10.1016/j.cell.2005.07.013. View

17.

Levy C, Verhoeyen E, Cosset F . Surface engineering of lentiviral vectors for gene transfer into gene therapy target cells. Curr Opin Pharmacol. 2015; 24:79-85. DOI: 10.1016/j.coph.2015.08.003. View

18.

Schenkwein D, Turkki V, Karkkainen H, Airenne K, Yla-Herttuala S . Production of HIV-1 integrase fusion protein-carrying lentiviral vectors for gene therapy and protein transduction. Hum Gene Ther. 2009; 21(5):589-602. DOI: 10.1089/hum.2009.051. View

19.

Gaj T, Guo J, Kato Y, Sirk S, Barbas 3rd C . Targeted gene knockout by direct delivery of zinc-finger nuclease proteins. Nat Methods. 2012; 9(8):805-7. PMC: 3424280. DOI: 10.1038/nmeth.2030. View

20.

Osborn M, Lonetree C, Webber B, Patel D, Dunmire S, McElroy A . CRISPR/Cas9 Targeted Gene Editing and Cellular Engineering in Fanconi Anemia. Stem Cells Dev. 2016; 25(20):1591-1603. PMC: 5035838. DOI: 10.1089/scd.2016.0149. View