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Spliceosome-mediated RNA trans-splicing as a tool for gene therapy

Nature Biotechnology volume 17, pages 246–252 (1999)Cite this article

A Corrigendum to this article was published on 01 March 2001

An Erratum to this article was published on 01 June 1999

Abstract

We have developed RNA molecules capable of effecting spliceosome-mediated RNA trans-splicing reactions with a target messenger RNA precursor (pre-mRNA). Targeted trans-splicing was demonstrated in a HeLa nuclear extract, cultured human cells, and H1299 human lung cancer tumors in athymic mice. Trans-splicing between a cancer-associated pre-mRNA encoding the β-subunit of human chorionic gonadotropin gene 6 and pre– trans-splicing molecule (PTM) RNA was accurate both in vitro and in vivo. Comparison of targeted versus nontargeted trans-splicing revealed a moderate level of specificity, which was improved by the addition of an internal inverted repeat encompassing the PTM splice site. Competition between cis- and trans-splicing demonstrated that cis-splicing can be inhibited by trans-splicing. RNA repair in a splicing model of a nonfunctional lacZ transcript was effected in cells by a PTM, which restored significant β-galactosidase activity. These observations suggest that spliceosome-mediated RNA trans-splicing may represent a general approach for reprogramming the sequence of targeted transcripts, providing a novel approach to gene therapy.

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Figure 1: Model PTM constructs and targeted trans-splicing strategy.
Figure 2: Improvement of trans-splicing specificity by the inclusion of a "safety" stem into PTMs.
Figure 3: In the presence of increasing PTM concentration, cis-splicing is inhibited in favor of trans-splicing.
Figure 4: PTMs are capable of trans-splicing in cultured human cancer cells.
Figure 5: Comparison of cis- and trans-splicing in vivo.
Figure 6: Trans-splicing can repair defective pre-mRNA with expression of functional protein.

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Acknowledgements

We thank Christopher D.J. Pearce and Bruce M. Cameron Jr. for enabling this project, Virginia G. Johnson (Food and Drug Administration) for providing DT-A clones, and Patrick M. O'Connor (National Cancer Institute) for useful discussions. Personal thanks (L.G.M.) to Carl R. Merril. This work was supported in full by Proteome Sciences plc.

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Authors and Affiliations

  1. Intronn LLC, Durham, 27701, NC

    M. Puttaraju, Sharon F. Jamison, S. Gary Mansfield, Mariano A. Garcia-Blanco & Lloyd G. Mitchell

  2. Proteome Sciences plc, Coveham House, Cobham, KT11 3EP, Surrey, UK

    M. Puttaraju, Sharon F. Jamison, S. Gary Mansfield, Mariano A. Garcia-Blanco & Lloyd G. Mitchell

  3. Duke University Medical Center, Durham, 27710, NC

    Mariano A. Garcia-Blanco

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  1. M. Puttaraju
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Correspondence to Mariano A. Garcia-Blanco or Lloyd G. Mitchell.

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Puttaraju, M., Jamison, S., Mansfield, S. et al. Spliceosome-mediated RNA trans-splicing as a tool for gene therapy . Nat Biotechnol 17, 246–252 (1999). https://doi.org/10.1038/6986

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