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Engineering the splice acceptor for improved gene expression and viral titer in an MLV-based retroviral vector

Abstract

We have recently developed a retroviral vector that contains a splice acceptor from the human EF1-α gene and drives a significantly higher level of gene expression than other well known murine leukemia virus-based vectors. However, one downside of this vector is that viral titer significantly varies depending on the packaging lines used. Results from Northern blot analysis indicated that in certain cell lines the genomic transcript containing the packaging signal sequence was too efficiently spliced to the subgenomic RNA, resulting in low levels of genomic RNA and thus leading to a low viral titer. We tested the possibility of overcoming this problem by introducing mutations around the splice acceptor sequence in such a way that a delicate balance was maintained between the splicing efficiency (which determines the level of gene expression) and the amount of genomic transcript (which influences viral titer). After mutational analysis, one such mutant was found to meet this requirement. The newly constructed vector containing the engineered splice acceptor could indeed drive higher levels of expression in many therapeutic genes than other control vectors, without significantly compromising viral titer.

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References

  1. Yu SS, Kim JM, Kim S . High efficiency retroviral vectors that contain no viral coding sequences. Gene Therapy 2000; 7: 797–804.

    CAS  Article  PubMed  Google Scholar 

  2. Wiley – The Journal of Gene Medicine Website (http://www.wiley.co.uk/genetherapy).

  3. Cavazzana-Calvo M et al. Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science 2000; 288: 669–672.

    CAS  Article  PubMed  Google Scholar 

  4. Boggs SS et al. Prolonged systemic expression of human IL-1 receptor antagonist (hIL-1ra) in mice reconstituted with hematopoietic cells transduced with a retrovirus carrying the hIL-1ra cDNA. Gene Therapy 1995; 2: 632–638.

    CAS  PubMed  Google Scholar 

  5. Bowtell DD, Cory S, Johnson GR, Gonda TJ . Comparison of expression in hemopoietic cells by retroviral vectors carrying two genes. J Virol 1988; 62: 2464–2473.

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Dranoff G et al. Vaccination with irradiated tumor cells engineered to secrete murine GM-CSF stimulates potent, specific and long lasting antitumor immunity. Proc Natl Acad Sci USA 1993; 90: 3539–3543.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. Jaffee EM et al. High efficiency gene transfer into primary human tumor explants without cell selection. Cancer Res 1993; 53 (10 Suppl): 2221–2226.

    CAS  PubMed  Google Scholar 

  8. Ohashi T et al. Efficient transfer and sustained high expression of the human glucocerebrosidase gene in mice and their functional macrophages following transplantation of bone marrow transduced by a retroviral vector. Proc Natl Acad Sci USA 1992; 89: 11332–11336.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. Palmer TD, Thompson AR, Miller AD . Production of human factor IX in animals by genetically modified skin fibroblasts: potential therapy for hemophilia B. Blood 1989; 73: 438–445.

    CAS  PubMed  Google Scholar 

  10. Miller AD, Roseman GJ . Improved retroviral vectors for gene transfer and expression. Biotechniques 1989; 7: 980–990.

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Miller AD, Trauber DR, Buttimore C . Factors involved in production of helper virus free retrovirus vectors. Somat Cell Mol Genet 1986; 12: 175–183.

    CAS  Article  PubMed  Google Scholar 

  12. Muenchau DD et al. Analysis of retroviral packaging lines for generation of replication-competent virus. Virology 1990; 176: 262–265.

    CAS  Article  PubMed  Google Scholar 

  13. Otto E et al. Characterization of a replication-competent retrovirus resulting from recombination of packaging and vector sequences. Hum Gene Ther 1994; 5: 567–575.

    CAS  Article  PubMed  Google Scholar 

  14. Padgett RA et al. Splicing of messenger RNA precursors. Annu Rev Biochem 1986; 55: 1119–1150 review).

    CAS  Article  PubMed  Google Scholar 

  15. Green MR . Biochemical mechanisms of constitutive and regulated pre-mRNA splicing. Annu Rev Cell Biol 1991; 7: 559–599 review).

    CAS  Article  PubMed  Google Scholar 

  16. Carothers AM, Urlaub G, Grunberger D, Chasin LA . Splicing mutants and their second-site suppressors at the dihydrofolate reductase locus in Chinese hamster ovary cells. Mol Cell Biol 1993; 13: 5085–5098.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. Zitvogel L et al. Construction and characterization of retroviral vectors expressing biologically active human interleukin-12. Hum Gene Ther 1994; 5: 1493–1506.

    CAS  Article  PubMed  Google Scholar 

  18. Keller EB, Noon WA . Intron splicing: a conserved internal signal in introns of animal pre-mRNAs. Proc Natl Acad Sci USA 1984; 81: 7417–7420.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. Madhani HD, Guthrie C . Dynamic RNA–RNA interactions in the spliceosome. Annu Rev Genet 1994; 28: 1–26 (review).

    CAS  Article  PubMed  Google Scholar 

  20. Reed R . Initial splice-site recognition and pairing during pre-mRNA splicing. Curr Opin Genet Dev 1996; 6: 215–220 (review).

    CAS  Article  PubMed  Google Scholar 

  21. Will CL, Luhrmann R . Protein functions in pre-mRNA splicing. Curr Opin Cell Biol 1997; 9: 320–328 (review).

    CAS  Article  PubMed  Google Scholar 

  22. Hornig H, Aebi M, Weissmann C . Effect of mutations at the lariat branch acceptor site on beta-globin pre-mRNA splicing in vitro. Nature 1986; 324: 589–591.

    CAS  Article  PubMed  Google Scholar 

  23. Macklin WB, Gardinier MV, King KD, Kampf K . An AG–GG transition at a splice site in the myelin proteolipid protein gene in jimpy mice results in the removal of an exon. FEBS Lett 1987; 223: 417–421 (letter).

    CAS  Article  PubMed  Google Scholar 

  24. Hedley ML, Forman J, Tucker PW . Mutation of 3′ splice sites in two different class I genes results in different usage of cryptic splice sites. J Immunol 1989; 143: 1018–1025.

    CAS  PubMed  Google Scholar 

  25. Yu SS et al. Construction of a retroviral vector production system with the minimum possibility of a homologous recombination. Gene Therapy 2003; 10: 706–711.

    CAS  Article  PubMed  Google Scholar 

  26. Palmiter RD et al. Heterologous introns can enhance expression of transgenes in mice. Proc Natl Acad Sci USA 1991; 88: 478–482.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  27. Luo MJ, Reed R . Splicing is required for rapid and efficient mRNA export in metazoans. Proc Natl Acad Sci USA 1999; 96: 14937–14942.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  28. Brinster RL et al. Introns increase transcriptional efficiency in transgenic mice. Proc Natl Acad Sci USA 1988; 85: 836–840.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. Choi T, Huang M, Gorman C, Jaenisch R . A generic intron increases gene expression in transgenic mice. Mol Cell Biol 1991; 11: 3070–3074.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. Krall WJ et al. Increased levels of spliced RNA account for augmented expression from the MFG retroviral vector in hematopoietic cells. Gene Therapy 1996; 3: 37–48.

    CAS  PubMed  Google Scholar 

  31. Hildinger M, Abel KL, Ostertag W, Baum C . Design of 5′ untranslated sequences in retroviral vectors developed for medical use. J Virol 1999; 73: 4083–4089.

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Ohashi T et al. Characterization of human glucocerebrosidase from different mutant alleles. J Biol Chem 1991; 266: 3661–3667.

    CAS  PubMed  Google Scholar 

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Acknowledgements

We are grateful to V Narry Kim for critical reading of this manuscript and helpful discussion. This work was supported in part by grants from the Korean Ministry of Commerce, Industry and Energy (Grant no. ND3-990-5411-031-1-3), the Ministry of Science and Technology (Grant no. MI-9808-00-0040) and IVI-Affiliated Lab Program (01-1-1).

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Lee, JT., Yu, S., Han, E. et al. Engineering the splice acceptor for improved gene expression and viral titer in an MLV-based retroviral vector. Gene Ther 11, 94–99 (2004). https://doi.org/10.1038/sj.gt.3302138

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  • DOI: https://doi.org/10.1038/sj.gt.3302138

Keywords

  • retroviral vector
  • splicing
  • engineered splice acceptor
  • level of gene expression

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