Skip to main content
PMC home page
The Journal of Experimental Medicine logoLink to The Journal of Experimental Medicine
. 1994 Feb 1;179(2):733–738. doi: 10.1084/jem.179.2.733

Suppression of human alpha-globin gene expression mediated by the recombinant adeno-associated virus 2-based antisense vectors

PMCID: PMC2191360  PMID: 8294880

Abstract

We sought to investigate the usefulness of the adeno-associated virus 2 (AAV)-based vectors to suppress the excess production of the human alpha-globin gene product towards developing a treatment modality for beta-thalassemia since accumulation of free alpha-globin reduces the lifespan of red blood cells in these patients. We constructed recombinant AAV virions containing the human alpha-globin gene sequences in antisense orientation driven by the herpesvirus thymidine kinase (TK) promoter, the SV40 early gene promoter, and the human alpha- globin gene promoter, respectively, as well as a bacterial gene for resistance to neomycin (neoR) as a selectable marker. These recombinant virions were used to infect a human erythroleukemia cell line (K562) that express high levels of alpha-globin mRNA. Clonal populations of neoR cells were obtained after selection with the drug G418, a neomycin analogue. Total genomic DNA samples isolated from these cells were analyzed on Southern blots to document stable integration of the transduced neo and alpha-globin genes. Total cellular RNA samples isolated from mock-infected and recombinant virus-infected cultures were also analyzed by Northern blots. Whereas the TK promoter-driven antisense alpha-globin sequences showed no inhibition of expression of the endogenous alpha-globin gene, the SV40 promoter and the alpha- globin gene promoter-driven antisense alpha-globin sequences suppressed the expression of this constitutively over-expressed gene by approximately 29 and 91%, respectively, at the transcriptional level. These studies suggest the feasibility of utilizing the AAV-based antisense gene transfer approach in the potential treatment of beta- thalassemia.

Full Text

The Full Text of this article is available as a PDF (785.3 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Berns K. I., Bohenzky R. A. Adeno-associated viruses: an update. Adv Virus Res. 1987;32:243–306. doi: 10.1016/s0065-3527(08)60479-0. [DOI] [PubMed] [Google Scholar]
  2. Blacklow N. R., Hoggan M. D., Sereno M. S., Brandt C. D., Kim H. W., Parrott R. H., Chanock R. M. A seroepidemiologic study of adenovirus-associated virus infection in infants and children. Am J Epidemiol. 1971 Oct;94(4):359–366. doi: 10.1093/oxfordjournals.aje.a121331. [DOI] [PubMed] [Google Scholar]
  3. Carter B. J. Adeno-associated virus vectors. Curr Opin Biotechnol. 1992 Oct;3(5):533–539. doi: 10.1016/0958-1669(92)90082-t. [DOI] [PubMed] [Google Scholar]
  4. Chatterjee S., Johnson P. R., Wong K. K., Jr Dual-target inhibition of HIV-1 in vitro by means of an adeno-associated virus antisense vector. Science. 1992 Nov 27;258(5087):1485–1488. doi: 10.1126/science.1359646. [DOI] [PubMed] [Google Scholar]
  5. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  6. Cukor G., Blacklow N. R., Kibrick S., Swan I. C. Effect of adeno-associated virus on cancer expression by herpesvirus-transformed hamster cells. J Natl Cancer Inst. 1975 Oct;55(4):957–959. doi: 10.1093/jnci/55.4.957. [DOI] [PubMed] [Google Scholar]
  7. Donahue R. E., Kessler S. W., Bodine D., McDonagh K., Dunbar C., Goodman S., Agricola B., Byrne E., Raffeld M., Moen R. Helper virus induced T cell lymphoma in nonhuman primates after retroviral mediated gene transfer. J Exp Med. 1992 Oct 1;176(4):1125–1135. doi: 10.1084/jem.176.4.1125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hermonat P. L. Inhibition of H-ras expression by the adeno-associated virus Rep78 transformation suppressor gene product. Cancer Res. 1991 Jul 1;51(13):3373–3377. [PubMed] [Google Scholar]
  9. Kotin R. M., Menninger J. C., Ward D. C., Berns K. I. Mapping and direct visualization of a region-specific viral DNA integration site on chromosome 19q13-qter. Genomics. 1991 Jul;10(3):831–834. doi: 10.1016/0888-7543(91)90470-y. [DOI] [PubMed] [Google Scholar]
  10. Kotin R. M., Siniscalco M., Samulski R. J., Zhu X. D., Hunter L., Laughlin C. A., McLaughlin S., Muzyczka N., Rocchi M., Berns K. I. Site-specific integration by adeno-associated virus. Proc Natl Acad Sci U S A. 1990 Mar;87(6):2211–2215. doi: 10.1073/pnas.87.6.2211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Muzyczka N. Use of adeno-associated virus as a general transduction vector for mammalian cells. Curr Top Microbiol Immunol. 1992;158:97–129. doi: 10.1007/978-3-642-75608-5_5. [DOI] [PubMed] [Google Scholar]
  12. Nahreini P., Larsen S. H., Srivastava A. Cloning and integration of DNA fragments in human cells via the inverted terminal repeats of the adeno-associated virus 2 genome. Gene. 1992 Oct 1;119(2):265–272. doi: 10.1016/0378-1119(92)90281-s. [DOI] [PubMed] [Google Scholar]
  13. Nahreini P., Srivastava A. Rescue and replication of the adeno-associated virus 2 genome in mortal and immortal human cells. Intervirology. 1989;30(2):74–85. doi: 10.1159/000150078. [DOI] [PubMed] [Google Scholar]
  14. Nahreini P., Srivastava A. Rescue of the adeno-associated virus 2 genome correlates with alterations in DNA-modifying enzymes in human cells. Intervirology. 1992;33(2):109–115. doi: 10.1159/000150239. [DOI] [PubMed] [Google Scholar]
  15. Nahreini P., Woody M. J., Zhou S. Z., Srivastava A. Versatile adeno-associated virus 2-based vectors for constructing recombinant virions. Gene. 1993 Feb 28;124(2):257–262. doi: 10.1016/0378-1119(93)90402-o. [DOI] [PubMed] [Google Scholar]
  16. Ostrove J. M., Duckworth D. H., Berns K. I. Inhibition of adenovirus-transformed cell oncogenicity by adeno-associated virus. Virology. 1981 Sep;113(2):521–533. doi: 10.1016/0042-6822(81)90180-x. [DOI] [PubMed] [Google Scholar]
  17. Samulski R. J. Adeno-associated virus: integration at a specific chromosomal locus. Curr Opin Genet Dev. 1993 Feb;3(1):74–80. doi: 10.1016/s0959-437x(05)80344-2. [DOI] [PubMed] [Google Scholar]
  18. Samulski R. J., Berns K. I., Tan M., Muzyczka N. Cloning of adeno-associated virus into pBR322: rescue of intact virus from the recombinant plasmid in human cells. Proc Natl Acad Sci U S A. 1982 Mar;79(6):2077–2081. doi: 10.1073/pnas.79.6.2077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Samulski R. J., Chang L. S., Shenk T. A recombinant plasmid from which an infectious adeno-associated virus genome can be excised in vitro and its use to study viral replication. J Virol. 1987 Oct;61(10):3096–3101. doi: 10.1128/jvi.61.10.3096-3101.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Samulski R. J., Chang L. S., Shenk T. Helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression. J Virol. 1989 Sep;63(9):3822–3828. doi: 10.1128/jvi.63.9.3822-3828.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Samulski R. J., Zhu X., Xiao X., Brook J. D., Housman D. E., Epstein N., Hunter L. A. Targeted integration of adeno-associated virus (AAV) into human chromosome 19. EMBO J. 1991 Dec;10(12):3941–3950. doi: 10.1002/j.1460-2075.1991.tb04964.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  23. Srivastava A., Lu L. Replication of B19 parvovirus in highly enriched hematopoietic progenitor cells from normal human bone marrow. J Virol. 1988 Aug;62(8):3059–3063. doi: 10.1128/jvi.62.8.3059-3063.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Srivastava A., Lusby E. W., Berns K. I. Nucleotide sequence and organization of the adeno-associated virus 2 genome. J Virol. 1983 Feb;45(2):555–564. doi: 10.1128/jvi.45.2.555-564.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Srivastava A. Replication of the adeno-associated virus DNA termini in vitro. Intervirology. 1987;27(3):138–147. doi: 10.1159/000149732. [DOI] [PubMed] [Google Scholar]
  26. Srivastava C. H., Samulski R. J., Lu L., Larsen S. H., Srivastava A. Construction of a recombinant human parvovirus B19: adeno-associated virus 2 (AAV) DNA inverted terminal repeats are functional in an AAV-B19 hybrid virus. Proc Natl Acad Sci U S A. 1989 Oct;86(20):8078–8082. doi: 10.1073/pnas.86.20.8078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Stockhaus J., Höfer M., Renger G., Westhoff P., Wydrzynski T., Willmitzer L. Anti-sense RNA efficiently inhibits formation of the 10 kd polypeptide of photosystem II in transgenic potato plants: analysis of the role of the 10 kd protein. EMBO J. 1990 Sep;9(9):3013–3021. doi: 10.1002/j.1460-2075.1990.tb07494.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Walsh C. E., Liu J. M., Xiao X., Young N. S., Nienhuis A. W., Samulski R. J. Regulated high level expression of a human gamma-globin gene introduced into erythroid cells by an adeno-associated virus vector. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):7257–7261. doi: 10.1073/pnas.89.15.7257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Zhou S. Z., Broxmeyer H. E., Cooper S., Harrington M. A., Srivastava A. Adeno-associated virus 2-mediated gene transfer in murine hematopoietic progenitor cells. Exp Hematol. 1993 Jul;21(7):928–933. [PubMed] [Google Scholar]

Articles from The Journal of Experimental Medicine are provided here courtesy of The Rockefeller University Press

RESOURCES