DOI: 10.1073/pnas.100082597. Preselection of retrovirally transduced bone marrow avoids subsequent stem cell gene silencing and age-dependent extinction of expression of human β-globin in engrafted mice

Preselection of retrovirally transduced bone marrow avoids subsequent stem cell gene silencing and age-dependent extinction of expression of human β-globin in engrafted mice

10.1073/pnas.100082597

Crossref journal-article

Proceedings of the National Academy of Sciences

Proceedings of the National Academy of Sciences (341)

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 β-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 β-globin gene in the erythroid lineage of transplanted mice. We observed that 100% of mice ( n = 7) engrafted with preselected cells concurrently expressed human β-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 β-locus control region hypersensitive site 2 alone, human β-globin mRNA expression levels ranged from 0.15% to 20% with human β-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.

Bibliography

Kalberer, C. P., Pawliuk, R., Imren, S., Bachelot, T., Takekoshi, K. J., Fabry, M., Eaves, C. J., London, I. M., Humphries, R. K., & Leboulch, P. (2000). Preselection of retrovirally transduced bone marrow avoids subsequent stem cell gene silencing and age-dependent extinction of expression of human Î²-globin in engrafted mice. Proceedings of the National Academy of Sciences, 97(10), 5411â5415.

Authors 10

Christian P. Kalberer (first)
Robert Pawliuk (additional)
Suzan Imren (additional)
Thomas Bachelot (additional)
Ken J. Takekoshi (additional)
Mary Fabry (additional)
Connie J. Eaves (additional)
Irving M. London (additional)
R. Keith Humphries (additional)
Philippe Leboulch (additional)

References 26 Referenced 70

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10.1016/0092-8674(87)90584-8
G Stamatoyannopoulos, A W Nienhuis The Molecular Basis of Blood Diseases, eds G Stamatoyannopoulos, A,W Nienhuis, P W Majerus, H Varmus (Saunders, Philadelphia), pp. 107–155 (1994). / The Molecular Basis of Blood Diseases by Stamatoyannopoulos G (1994)
10.1002/j.1460-2075.1994.tb06605.x
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10.1182/blood.V93.12.4059.412k40_4059_4070

Dates

Type	When
Created	23 years, 1 month ago (July 26, 2002, 10:38 a.m.)
Deposited	3 years, 4 months ago (April 12, 2022, 6:53 p.m.)
Indexed	1 year, 7 months ago (Jan. 9, 2024, 7:08 p.m.)
Issued	25 years, 4 months ago (May 2, 2000)
Published	25 years, 4 months ago (May 2, 2000)
Published Online	25 years, 4 months ago (May 2, 2000)
Published Print	25 years, 3 months ago (May 9, 2000)

Funders 0

None

BibTeX

@article{Kalberer_2000, title={Preselection of retrovirally transduced bone marrow avoids subsequent stem cell gene silencing and age-dependent extinction of expression of human β-globin in engrafted mice}, volume={97}, ISSN={1091-6490}, url={http://dx.doi.org/10.1073/pnas.100082597}, DOI={10.1073/pnas.100082597}, number={10}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Kalberer, Christian P. and Pawliuk, Robert and Imren, Suzan and Bachelot, Thomas and Takekoshi, Ken J. and Fabry, Mary and Eaves, Connie J. and London, Irving M. and Humphries, R. Keith and Leboulch, Philippe}, year={2000}, month=may, pages={5411–5415} }

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  "abstract": "<jats:p>\n            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 \u03b2-globin gene, even when cis-linked to locus control region derivatives. Here, we have investigated whether\n            <jats:italic>ex vivo</jats:italic>\n            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 \u03b2-globin gene in the erythroid lineage of transplanted mice. We observed that 100% of mice (\n            <jats:italic>n</jats:italic>\n            = 7) engrafted with preselected cells concurrently expressed human \u03b2-globin and the green fluorescent protein in 20\u201395% 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 \u03b2-locus control region hypersensitive site 2 alone, human \u03b2-globin mRNA expression levels ranged from 0.15% to 20% with human \u03b2-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,\n            <jats:italic>in vivo</jats:italic>\n            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.\n          </jats:p>",
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