Conditional Splicing System for Tight Control of Viral Overlapping Genes

Overview

Journal J Virol

Publisher American Society for Microbiology

Date 2024 Mar 6

PMID 38446633

Authors

Qing Yang

Jinlin Wang

Zhiwei Chen

Affiliations

Soon will be listed here.

Abstract

Importance: Regulating overlapping genes is vital for gene therapy and vaccine development using viral vectors. The regulation of overlapping genes presents challenges, including cytotoxicity and impacts on vector capacity and genome stability, which restrict stable packaging cell line development and broad application. To address these challenges, we present a "loxp-splice-loxp"-based conditional splicing system, offering a novel solution for conditional expression of overlapping genes and stable cell line establishment. This system may also regulate other cytotoxic genes, representing a significant advancement in cell engineering and gene therapy as well as biomass production.

References

Penaud-Budloo M, Francois A, Clement N, Ayuso E . Pharmacology of Recombinant Adeno-associated Virus Production. Mol Ther Methods Clin Dev. 2018; 8:166-180. PMC: 5908265. DOI: 10.1016/j.omtm.2018.01.002. View

Palmer D, Ng P . Improved system for helper-dependent adenoviral vector production. Mol Ther. 2003; 8(5):846-52. DOI: 10.1016/j.ymthe.2003.08.014. View

Lasaro M, Ertl H . New insights on adenovirus as vaccine vectors. Mol Ther. 2009; 17(8):1333-9. PMC: 2835230. DOI: 10.1038/mt.2009.130. View

Yuan Z, Qiao C, Hu P, Li J, Xiao X . A versatile adeno-associated virus vector producer cell line method for scalable vector production of different serotypes. Hum Gene Ther. 2010; 22(5):613-24. PMC: 3081441. DOI: 10.1089/hum.2010.241. View

Jaganathan K, Kyriazopoulou Panagiotopoulou S, McRae J, Fazel Darbandi S, Knowles D, Li Y . Predicting Splicing from Primary Sequence with Deep Learning. Cell. 2019; 176(3):535-548.e24. DOI: 10.1016/j.cell.2018.12.015. View

Jackson R, Kroehling L, Khitun A, Bailis W, Jarret A, York A . The translation of non-canonical open reading frames controls mucosal immunity. Nature. 2018; 564(7736):434-438. PMC: 6939389. DOI: 10.1038/s41586-018-0794-7. View

Paz S, Krainer A, Caputi M . HIV-1 transcription is regulated by splicing factor SRSF1. Nucleic Acids Res. 2014; 42(22):13812-23. PMC: 4267630. DOI: 10.1093/nar/gku1170. View

OGorman S, Fox D, Wahl G . Recombinase-mediated gene activation and site-specific integration in mammalian cells. Science. 1991; 251(4999):1351-5. DOI: 10.1126/science.1900642. View

Richter S, Belanger F, Zheng P, Rana T . Dynamics of nascent mRNA folding and RNA-protein interactions: an alternative TAR RNA structure is involved in the control of HIV-1 mRNA transcription. Nucleic Acids Res. 2006; 34(15):4278-92. PMC: 1616951. DOI: 10.1093/nar/gkl499. View

10.

Sauer B, Henderson N . Cre-stimulated recombination at loxP-containing DNA sequences placed into the mammalian genome. Nucleic Acids Res. 1989; 17(1):147-61. PMC: 331541. DOI: 10.1093/nar/17.1.147. View

11.

Forrest A, Kawaji H, Rehli M, Baillie J, de Hoon M, Haberle V . A promoter-level mammalian expression atlas. Nature. 2014; 507(7493):462-70. PMC: 4529748. DOI: 10.1038/nature13182. View

12.

Lemay J, Bachand F . Fail-safe transcription termination: Because one is never enough. RNA Biol. 2015; 12(9):927-32. PMC: 4615224. DOI: 10.1080/15476286.2015.1073433. View

13.

Bapst A, Dahl S, Knopfel T, Wenger R . Cre-mediated, loxP independent sequential recombination of a tripartite transcriptional stop cassette allows for partial read-through transcription. Biochim Biophys Acta Gene Regul Mech. 2020; 1863(8):194568. DOI: 10.1016/j.bbagrm.2020.194568. View

14.

Qiao C, Li J, Skold A, Zhang X, Xiao X . Feasibility of generating adeno-associated virus packaging cell lines containing inducible adenovirus helper genes. J Virol. 2002; 76(4):1904-13. PMC: 135905. DOI: 10.1128/jvi.76.4.1904-1913.2002. View

15.

Schlub T, Holmes E . Properties and abundance of overlapping genes in viruses. Virus Evol. 2020; 6(1):veaa009. PMC: 7017920. DOI: 10.1093/ve/veaa009. View

16.

Yang Q, Chen F, Trempe J . Characterization of cell lines that inducibly express the adeno-associated virus Rep proteins. J Virol. 1994; 68(8):4847-56. PMC: 236424. DOI: 10.1128/JVI.68.8.4847-4856.1994. View

17.

Robert M, Chahal P, Audy A, Kamen A, Gilbert R, Gaillet B . Manufacturing of recombinant adeno-associated viruses using mammalian expression platforms. Biotechnol J. 2017; 12(3). DOI: 10.1002/biot.201600193. View

18.

Chen J, Brunner A, Cogan J, Nunez J, Fields A, Adamson B . Pervasive functional translation of noncanonical human open reading frames. Science. 2020; 367(6482):1140-1146. PMC: 7289059. DOI: 10.1126/science.aay0262. View

19.

Berg K, Schafer V, Bartnicki N, Eggenschwiler R, Cantz T, Stitz J . Rapid establishment of stable retroviral packaging cells and recombinant susceptible target cell lines employing novel transposon vectors derived from Sleeping Beauty. Virology. 2019; 531:40-47. DOI: 10.1016/j.virol.2019.02.014. View

20.

Shaffer J, Greenwald I . Floxed exon (Flexon): A flexibly positioned stop cassette for recombinase-mediated conditional gene expression. Proc Natl Acad Sci U S A. 2022; 119(3). PMC: 8784106. DOI: 10.1073/pnas.2117451119. View