Preselection of Retrovirally Transduced Bone Marrow Avoids Subsequent Stem Cell Gene Silencing and Age-dependent Extinction of Expression of Human Beta-globin in Engrafted Mice

Overview

Journal Proc Natl Acad Sci U S A

Specialty Science

Date 2000 May 3

PMID 10792053

Citations 20

Authors

C P Kalberer

R Pawliuk

S Imren

T Bachelot

K J Takekoshi

M Fabry

C J Eaves

I M LONDON

R K Humphries

P Leboulch

Affiliations

Soon will be listed here.

Abstract

Transcriptional silencing of genes transferred into hematopoietic stem cells poses one of the most significant challenges to the success of gene therapy. If the transferred gene is not completely silenced, a progressive decline in gene expression as the mice age often is encountered. These phenomena were observed to various degrees in mouse transplant experiments using retroviral vectors containing a human beta-globin gene, even when cis-linked to locus control region derivatives. Here, we have investigated whether ex vivo preselection of retrovirally transduced stem cells on the basis of expression of the green fluorescent protein driven by the CpG island phosphoglycerate kinase promoter can ensure subsequent long-term expression of a cis-linked beta-globin gene in the erythroid lineage of transplanted mice. We observed that 100% of mice (n = 7) engrafted with preselected cells concurrently expressed human beta-globin and the green fluorescent protein in 20-95% of their RBC for up to 9.5 mo posttransplantation, the longest time point assessed. This expression pattern was successfully transferred to secondary transplant recipients. In the presence of beta-locus control region hypersensitive site 2 alone, human beta-globin mRNA expression levels ranged from 0.15% to 20% with human beta-globin chains detected by HPLC. Neither the proportion of positive blood cells nor the average expression levels declined with time in transplanted recipients. Although suboptimal expression levels and heterocellular position effects persisted, in vivo stem cell gene silencing and age-dependent extinction of expression were avoided. These findings support the further investigation of this type of vector for the gene therapy of human hemoglobinopathies.

Citing Articles

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References

Tyler J, Kadonaga J . The "dark side" of chromatin remodeling: repressive effects on transcription. Cell. 1999; 99(5):443-6. DOI: 10.1016/s0092-8674(00)81530-5. View

Pawliuk R, Bachelot T, Wise R, Leboulch P . Long-term cure of the photosensitivity of murine erythropoietic protoporphyria by preselective gene therapy. Nat Med. 1999; 5(7):768-73. DOI: 10.1038/10488. View

Bird A, Wolffe A . Methylation-induced repression--belts, braces, and chromatin. Cell. 1999; 99(5):451-4. DOI: 10.1016/s0092-8674(00)81532-9. View

Sun F, Elgin S . Putting boundaries on silence. Cell. 1999; 99(5):459-62. DOI: 10.1016/s0092-8674(00)81534-2. View

Tuan D, SOLOMON W, Li Q, LONDON I . The "beta-like-globin" gene domain in human erythroid cells. Proc Natl Acad Sci U S A. 1985; 82(19):6384-8. PMC: 390720. DOI: 10.1073/pnas.82.19.6384. View

Grosveld F, Van Assendelft G, Greaves D, Kollias G . Position-independent, high-level expression of the human beta-globin gene in transgenic mice. Cell. 1987; 51(6):975-85. DOI: 10.1016/0092-8674(87)90584-8. View

Markowitz D, Goff S, Bank A . A safe packaging line for gene transfer: separating viral genes on two different plasmids. J Virol. 1988; 62(4):1120-4. PMC: 253118. DOI: 10.1128/JVI.62.4.1120-1124.1988. View

Markowitz D, Goff S, Bank A . Construction and use of a safe and efficient amphotropic packaging cell line. Virology. 1988; 167(2):400-6. View

Tuan D, Solomon W, LONDON I, Lee D . An erythroid-specific, developmental-stage-independent enhancer far upstream of the human "beta-like globin" genes. Proc Natl Acad Sci U S A. 1989; 86(8):2554-8. PMC: 286955. DOI: 10.1073/pnas.86.8.2554. View

10.

Levitt N, Briggs D, Gil A, Proudfoot N . Definition of an efficient synthetic poly(A) site. Genes Dev. 1989; 3(7):1019-25. DOI: 10.1101/gad.3.7.1019. View

11.

Razin A, Cedar H . DNA methylation and gene expression. Microbiol Rev. 1991; 55(3):451-8. PMC: 372829. DOI: 10.1128/mr.55.3.451-458.1991. View

12.

Kafri T, Ariel M, Brandeis M, Shemer R, Urven L, McCarrey J . Developmental pattern of gene-specific DNA methylation in the mouse embryo and germ line. Genes Dev. 1992; 6(5):705-14. DOI: 10.1101/gad.6.5.705. View

13.

Fabry M, Costantini F, Pachnis A, Suzuka S, BANK N, AYNEDJIAN H . High expression of human beta S- and alpha-globins in transgenic mice: erythrocyte abnormalities, organ damage, and the effect of hypoxia. Proc Natl Acad Sci U S A. 1992; 89(24):12155-9. PMC: 50717. DOI: 10.1073/pnas.89.24.12155. View

14.

Leboulch P, Huang G, Humphries R, Oh Y, Eaves C, Tuan D . Mutagenesis of retroviral vectors transducing human beta-globin gene and beta-globin locus control region derivatives results in stable transmission of an active transcriptional structure. EMBO J. 1994; 13(13):3065-76. PMC: 395197. DOI: 10.1002/j.1460-2075.1994.tb06605.x. View

15.

Pawliuk R, Kay R, Lansdorp P, Humphries R . Selection of retrovirally transduced hematopoietic cells using CD24 as a marker of gene transfer. Blood. 1994; 84(9):2868-77. View

16.

Huang Z, Yen T . Role of the hepatitis B virus posttranscriptional regulatory element in export of intronless transcripts. Mol Cell Biol. 1995; 15(7):3864-9. PMC: 230626. DOI: 10.1128/MCB.15.7.3864. View

17.

Hawley R, Lieu F, Fong A, Hawley T . Versatile retroviral vectors for potential use in gene therapy. Gene Ther. 1994; 1(2):136-8. View

18.

Ellis J, Pasceri P, Tan-Un K, Wu X, Harper A, Fraser P . Evaluation of beta-globin gene therapy constructs in single copy transgenic mice. Nucleic Acids Res. 1997; 25(6):1296-302. PMC: 146564. DOI: 10.1093/nar/25.6.1296. View

19.

Chen W, Bailey E, McCune S, Dong J, Townes T . Reactivation of silenced, virally transduced genes by inhibitors of histone deacetylase. Proc Natl Acad Sci U S A. 1997; 94(11):5798-803. PMC: 20860. DOI: 10.1073/pnas.94.11.5798. View

20.

Pawliuk R, Eaves C, Humphries R . Sustained high-level reconstitution of the hematopoietic system by preselected hematopoietic cells expressing a transduced cell-surface antigen. Hum Gene Ther. 1997; 8(13):1595-604. DOI: 10.1089/hum.1997.8.13-1595. View