Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

A small regulatory element from chromosome 19 enhances liver-specific gene expression

Abstract

Tissue-specific promoters for gene therapy are typically too big for adeno-associated virus (AAV) vectors; thus, the exploration of small effective non-viral regulatory elements is of particular interest. Wild-type AAV can specifically integrate into a region on human chromosome 19 termed AAVS1. Earlier work has determined that a 347 bp fragment (Chr19) of AAVS1 has promoter and transcriptional enhancer activities. In this study, we further characterized this genetic regulation and investigated its application to AAV gene therapy in vitro and in vivo. The Chr19 347 bp fragment was dissected into three regulatory elements in human embryonic kidney cells: (i) TATA-independent promoter activity distributed throughout the fragment regardless of orientation, (ii) an orientation-dependent insulator function near the 5′ end and (iii) a 107 bp enhancer region near the 3′ end. The small enhancer region, coupled to the mini-CMV promoter, was used to drive the expression of several reporters following transduction by AAV2. In vivo data demonstrated enhanced transgene expression from the Chr19-mini-CMV promoter cassette after tail vein injection primarily in the liver at levels comparable to the chicken β-actin promoter and higher than the liver-specific TTR promoter (>2-fold). However, we did not observe this increase after muscle injection, suggesting tissue-specific enhancement. All of the results support identification of a small DNA fragment (347 bp) from AAV Chr19 integration site capable of providing efficient and enhanced liver-specific transcription when used in recombinant AAV vectors.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6

Similar content being viewed by others

References

  1. Li C, Bowles DE, van Dyke T, Samulski RJ . Adeno-associated virus vectors: potential applications for cancer gene therapy. Cancer Gene Ther 2005; 12: 913–925.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Warrington Jr KH, Herzog RW . Treatment of human disease by adeno-associated viral gene transfer. Hum Genet 2006; 119: 571–603.

    Article  CAS  PubMed  Google Scholar 

  3. Fu H, Muenzer J, Samulski RJ, Breese G, Sifford J, Zeng X et al. Self-complementary adeno-associated virus serotype 2 vector: global distribution and broad dispersion of AAV-mediated transgene expression in mouse brain. Mol Ther 2003; 8: 911–917.

    Article  CAS  PubMed  Google Scholar 

  4. McCarty DM, Fu H, Monahan PE, Toulson CE, Naik P, Samulski RJ . Adeno-associated virus terminal repeat (TR) mutant generates self-complementary vectors to overcome the rate-limiting step to transduction in vivo. Gene Therapy 2003; 10: 2112–2118.

    Article  CAS  PubMed  Google Scholar 

  5. McCarty DM, Monahan PE, Samulski RJ . Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis. Gene Therapy 2001; 8: 1248–1254.

    Article  CAS  PubMed  Google Scholar 

  6. Wang Z, Ma HI, Li J, Sun L, Zhang J, Xiao X . Rapid and highly efficient transduction by double-stranded adeno-associated virus vectors in vitro and in vivo. Gene Therapy 2003; 10: 2105–2111.

    Article  CAS  PubMed  Google Scholar 

  7. Kotin RM, Linden RM, Berns KI . Characterization of a preferred site on human chromosome 19q for integration of adeno-associated virus DNA by non-homologous recombination. EMBO J 1992; 11: 5071–5078.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Lamartina S, Sporeno E, Fattori E, Toniatti C . Characteristics of the adeno-associated virus preintegration site in human chromosome 19: open chromatin conformation and transcription-competent environment. J Virol 2000; 74: 7671–7677.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Ogata T, Kozuka T, Kanda T . Identification of an insulator in AAVS1, a preferred region for integration of adeno-associated virus DNA. J Virol 2003; 77: 9000–9007.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Tan I, Ng CH, Lim L, Leung T . Phosphorylation of a novel myosin binding subunit of protein phosphatase 1 reveals a conserved mechanism in the regulation of actin cytoskeleton. J Biol Chem 2001; 276: 21209–21216.

    Article  CAS  PubMed  Google Scholar 

  11. Flotte TR, Solow R, Owens RA, Afione S, Zeitlin PL, Carter BJ . Gene expression from adeno-associated virus vectors in airway epithelial cells. Am J Respir Cell Mol Biol 1992; 7: 349–356.

    Article  CAS  PubMed  Google Scholar 

  12. Guo ZS, Wang LH, Eisensmith RC, Woo SL . Evaluation of promoter strength for hepatic gene expression in vivo following adenovirus-mediated gene transfer. Gene Therapy 1996; 3: 802–810.

    CAS  PubMed  Google Scholar 

  13. Prosch S, Stein J, Staak K, Liebenthal C, Volk HD, Kruger DH . Inactivation of the very strong HCMV immediate early promoter by DNA CpG methylation in vitro. Biol Chem Hoppe Seyler 1996; 377: 195–201.

    Article  CAS  PubMed  Google Scholar 

  14. Loser P, Jennings GS, Strauss M, Sandig V . Reactivation of the previously silenced cytomegalovirus major immediate-early promoter in the mouse liver: involvement of NFkappaB. J Virol 1998; 72: 180–190.

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Costa RH, Lai E, Darnell Jr JE . Transcriptional control of the mouse prealbumin (transthyretin) gene: both promoter sequences and a distinct enhancer are cell specific. Mol Cell Biol 1986; 6: 4697–4708.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Bartlett JS, Sethna M, Ramamurthy L, Gowen SA, Samulski RJ, Marzluff WF . Efficient expression of protein coding genes from the murine U1 small nuclear RNA promoters. Proc Natl Acad Sci USA 1996; 93: 8852–8857.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Wu Z, Sun J, Zhang T, Yin C, Yin F, Van Dyke T et al. Optimization of self-complementary AAV vectors for liver-directed expression results in sustained correction of Hemophilia B at low vector dose. Mol Ther 2007; 16: 280–289.

    Article  PubMed  Google Scholar 

  18. Wang Z, Zhu T, Qiao C, Zhou L, Wang B, Zhang J et al. Adeno-associated virus serotype 8 efficiently delivers genes to muscle and heart. Nat Biotechnol 2005; 23: 321–328.

    Article  CAS  PubMed  Google Scholar 

  19. Sun B, Zhang H, Franco LM, Young SP, Schneider A, Bird A et al. Efficacy of an adeno-associated virus 8-pseudotyped vector in glycogen storage disease type II. Mol Ther 2005; 11: 57–65.

    Article  CAS  PubMed  Google Scholar 

  20. Larsen F, Gundersen G, Lopez R, Prydz H . CpG islands as gene markers in the human genome. Genomics 1992; 13: 1095–1107.

    Article  CAS  PubMed  Google Scholar 

  21. Ponger L, Duret L, Mouchiroud D . Determinants of CpG islands: expression in early embryo and isochore structure. Genome Res 2001; 11: 1854–1860.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Yamashita R, Suzuki Y, Sugano S, Nakai K . Genome-wide analysis reveals strong correlation between CpG islands with nearby transcription start sites of genes and their tissue specificity. Gene 2005; 350: 129–136.

    Article  CAS  PubMed  Google Scholar 

  23. Gaszner M, Felsenfeld G . Insulators: exploiting transcriptional and epigenetic mechanisms. Nat Rev Genet 2006; 7: 703–713.

    Article  CAS  PubMed  Google Scholar 

  24. Xiao X, Li J, Samulski RJ . Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus. J Virol 1998; 72: 2224–2232.

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Li C, Hirsch M, Asokan A, Zeithaml B, Ma H, Kafri T et al. Adeno-associated virus type 2 (AAV2) capsid-specific cytotoxic T lymphocytes eliminate only vector-transduced cells coexpressing the AAV2 capsid in vivo. J Virol 2007; 81: 7540–7547.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by the NIH research grants 5P01GM059299, 5P01HL066973 and 2P01HL051818, and a research grant from Alpha-1 Foundation.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to R J Samulski.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Li, C., Hirsch, M., Carter, P. et al. A small regulatory element from chromosome 19 enhances liver-specific gene expression. Gene Ther 16, 43–51 (2009). https://doi.org/10.1038/gt.2008.134

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/gt.2008.134

Keywords

This article is cited by

Search

Quick links