Design of retrovirus vectors for transfer and expression of the human beta-globin gene

Regulated expression of the human beta-globin gene has been demonstrated in cultured murine erythroleukemia cells and in mice after retrovirus-mediated gene transfer. However, the low titer of recombinant viruses described to date results in relatively inefficient gene transfer, which limits their usefulness for animal studies and for potential gene therapy in humans for diseases involving defective beta-globin genes. We found regions that interfered with virus production within intron 2 of the beta-globin gene and on both sides of the gene. The flanking regions could be removed, but intron 2 was required for beta-globin expression. Inclusion of beta-globin introns necessitates an antisense orientation of the gene within the retrovirus vector. However, we found no effect of the antisense beta-globin transcription on virus production. A region downstream of the beta-globin gene that stimulates expression of the gene in transgenic mice was included in the viruses without detrimental effects on virus titer. Virus titers of over 10(6) CFU/ml were obtained with the final vector design, which retained the ability to direct regulated expression of human beta-globin in murine erythroleukemia cells. The vector also allowed transfer and expression of the human beta-globin gene in hematopoietic cells (CFU-S cells) in mice.

[1]  M. Bender,et al.  Expression of the human beta-globin gene after retroviral transfer into murine erythroleukemia cells and human BFU-E cells , 1988, Molecular and cellular biology.

[2]  R. Mulligan,et al.  Lineage-specific expression of a human β-globin gene in murine bone marrow transplant recipients reconstituted with retrovirus-transduced stem cells , 1988, Nature.

[3]  G. Kollias,et al.  Position-independent, high-level expression of the human β-globin gene in transgenic mice , 1987, Cell.

[4]  F. Costantini,et al.  A 3' enhancer contributes to the stage-specific expression of the human beta-globin gene. , 1987, Genes & development.

[5]  G. Kollias,et al.  The human β-globin gene contains a downstream developmental specific enhancer , 1987 .

[6]  M. Bender,et al.  Evidence that the packaging signal of Moloney murine leukemia virus extends into the gag region , 1987, Journal of virology.

[7]  Armentano,et al.  Effect of internal viral sequences on the utility of retroviral vectors , 1987, Journal of virology.

[8]  G. Stamatoyannopoulos,et al.  Retroviral-mediated transfer of genomic globin genes leads to regulated production of RNA and protein. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[9]  R. Mulligan,et al.  Regulated expression of a complete human beta-globin gene encoded by a transmissible retrovirus vector , 1987, Molecular and cellular biology.

[10]  R. Humphries,et al.  Gene transfer to primary normal and malignant human hemopoietic progenitors using recombinant retroviruses. , 1987, Blood.

[11]  A. Miller,et al.  Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production , 1986, Molecular and cellular biology.

[12]  A. Miller,et al.  Retrovirus-mediated transfer and expression of drug resistance genes in human haematopoietic progenitor cells , 1986, Nature.

[13]  A. Miller,et al.  Factors involved in production of helper virus-free retrovirus vectors , 1986, Somatic cell and molecular genetics.

[14]  H. Temin.  Retrovirus Vectors for Gene Transfer: Efficient Integration into and Expression of Exogenous DNA in Vertebrate Cell Genomes , 1986 .

[15]  M. Busslinger,et al.  Transcription termination and 3′ processing: the end is in site! , 1985, Cell.

[16]  I. Verma,et al.  Generation of helper-free amphotropic retroviruses that transduce a dominant-acting, methotrexate-resistant dihydrofolate reductase gene , 1985, Molecular and cellular biology.

[17]  R. Evans,et al.  Infectious and selectable retrovirus containing an inducible rat growth hormone minigene. , 1984, Science.

[18]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[19]  D. Baltimore,et al.  Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus , 1983, Cell.

[20]  G. Stamatoyannopoulos,et al.  Monoclonal antibodies specific for globin chains. , 1983, Blood.

[21]  B. Reiss,et al.  Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5. , 1982, Gene.

[22]  W. Scher,et al.  Hemoglobin synthesis in murine virus-induced leukemic cells in vitro: stimulation of erythroid differentiation by dimethyl sulfoxide. , 1971, Proceedings of the National Academy of Sciences of the United States of America.

[23]  J. Hartley,et al.  Plaque assay techniques for murine leukemia viruses. , 1970, Virology.