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Integration-specific In Vitro Evaluation of Lentivirally Transduced Rhesus CD34(+) Cells Correlates With In Vivo Vector Copy Number

Hematopoietic stem cell (HSC) gene therapy using integrating vectors has a potential leukemogenic risk due to insertional mutagenesis. To reduce this risk, a limitation of ≤2 average vector copy number (VCN) per cell is generally accepted. We developed an assay for VCN among transduced CD34(+) cells...

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Autores principales: Uchida, Naoya, Evans, Molly E, Hsieh, Matthew M, Bonifacino, Aylin C, Krouse, Allen E, Metzger, Mark E, Sellers, Stephanie E, Dunbar, Cynthia E, Donahue, Robert E, Tisdale, John F
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4098567/
https://www.ncbi.nlm.nih.gov/pubmed/24045711
http://dx.doi.org/10.1038/mtna.2013.49
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author Uchida, Naoya
Evans, Molly E
Hsieh, Matthew M
Bonifacino, Aylin C
Krouse, Allen E
Metzger, Mark E
Sellers, Stephanie E
Dunbar, Cynthia E
Donahue, Robert E
Tisdale, John F
author_facet Uchida, Naoya
Evans, Molly E
Hsieh, Matthew M
Bonifacino, Aylin C
Krouse, Allen E
Metzger, Mark E
Sellers, Stephanie E
Dunbar, Cynthia E
Donahue, Robert E
Tisdale, John F
author_sort Uchida, Naoya
collection PubMed
description Hematopoietic stem cell (HSC) gene therapy using integrating vectors has a potential leukemogenic risk due to insertional mutagenesis. To reduce this risk, a limitation of ≤2 average vector copy number (VCN) per cell is generally accepted. We developed an assay for VCN among transduced CD34(+) cells that reliably predicts in vivo VCN in 16 rhesus recipients of CD34(+) cells transduced with a green fluorescent protein (GFP) (or yellow fluorescent protein (YFP))-encoding lentiviral vector. Using GFP (or YFP)-specific probe/primers by real-time PCR, VCN among transduced CD34(+) cells had no correlation with VCN among granulocytes or lymphocytes in vivo assayed 6 months post-transplantation. This was a likely result of residual plasmids present in the vector preparation. We then designed self-inactivating long terminal repeat (SIN-LTR)-specific probe/primers, which detect only integrated provirus. Evaluation with SIN-LTR probe/primers resulted in a positive correlation of VCN among transduced CD34(+) cells with granulocytes and lymphocytes in vivo. The transduced CD34(+) cells had higher VCN (25.1 ± 5.6) as compared with granulocytes (2.8 ± 1) and lymphocytes (2.4 ± 0.7). In summary, an integrated provirus-specific real-time PCR system demonstrated nine- to tenfold higher VCN in transduced CD34(+) cells in vitro, as compared with VCN in vivo. Therefore, the restriction of ≤2 VCN before infusion might unnecessarily limit gene transfer efficacy.
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spelling pubmed-40985672014-07-17 Integration-specific In Vitro Evaluation of Lentivirally Transduced Rhesus CD34(+) Cells Correlates With In Vivo Vector Copy Number Uchida, Naoya Evans, Molly E Hsieh, Matthew M Bonifacino, Aylin C Krouse, Allen E Metzger, Mark E Sellers, Stephanie E Dunbar, Cynthia E Donahue, Robert E Tisdale, John F Mol Ther Nucleic Acids Methods - Original Article Hematopoietic stem cell (HSC) gene therapy using integrating vectors has a potential leukemogenic risk due to insertional mutagenesis. To reduce this risk, a limitation of ≤2 average vector copy number (VCN) per cell is generally accepted. We developed an assay for VCN among transduced CD34(+) cells that reliably predicts in vivo VCN in 16 rhesus recipients of CD34(+) cells transduced with a green fluorescent protein (GFP) (or yellow fluorescent protein (YFP))-encoding lentiviral vector. Using GFP (or YFP)-specific probe/primers by real-time PCR, VCN among transduced CD34(+) cells had no correlation with VCN among granulocytes or lymphocytes in vivo assayed 6 months post-transplantation. This was a likely result of residual plasmids present in the vector preparation. We then designed self-inactivating long terminal repeat (SIN-LTR)-specific probe/primers, which detect only integrated provirus. Evaluation with SIN-LTR probe/primers resulted in a positive correlation of VCN among transduced CD34(+) cells with granulocytes and lymphocytes in vivo. The transduced CD34(+) cells had higher VCN (25.1 ± 5.6) as compared with granulocytes (2.8 ± 1) and lymphocytes (2.4 ± 0.7). In summary, an integrated provirus-specific real-time PCR system demonstrated nine- to tenfold higher VCN in transduced CD34(+) cells in vitro, as compared with VCN in vivo. Therefore, the restriction of ≤2 VCN before infusion might unnecessarily limit gene transfer efficacy. Nature Publishing Group 2013-09 2013-09-17 /pmc/articles/PMC4098567/ /pubmed/24045711 http://dx.doi.org/10.1038/mtna.2013.49 Text en Copyright © 2013 American Society of Gene & Cell Therapy http://creativecommons.org/licenses/by-nc-nd/3.0/ Molecular Therapy-Nucleic Acids is an open-access journal published by Nature Publishing Group. This work is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 3.0 License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/3.0/
spellingShingle Methods - Original Article
Uchida, Naoya
Evans, Molly E
Hsieh, Matthew M
Bonifacino, Aylin C
Krouse, Allen E
Metzger, Mark E
Sellers, Stephanie E
Dunbar, Cynthia E
Donahue, Robert E
Tisdale, John F
Integration-specific In Vitro Evaluation of Lentivirally Transduced Rhesus CD34(+) Cells Correlates With In Vivo Vector Copy Number
title Integration-specific In Vitro Evaluation of Lentivirally Transduced Rhesus CD34(+) Cells Correlates With In Vivo Vector Copy Number
title_full Integration-specific In Vitro Evaluation of Lentivirally Transduced Rhesus CD34(+) Cells Correlates With In Vivo Vector Copy Number
title_fullStr Integration-specific In Vitro Evaluation of Lentivirally Transduced Rhesus CD34(+) Cells Correlates With In Vivo Vector Copy Number
title_full_unstemmed Integration-specific In Vitro Evaluation of Lentivirally Transduced Rhesus CD34(+) Cells Correlates With In Vivo Vector Copy Number
title_short Integration-specific In Vitro Evaluation of Lentivirally Transduced Rhesus CD34(+) Cells Correlates With In Vivo Vector Copy Number
title_sort integration-specific in vitro evaluation of lentivirally transduced rhesus cd34(+) cells correlates with in vivo vector copy number
topic Methods - Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4098567/
https://www.ncbi.nlm.nih.gov/pubmed/24045711
http://dx.doi.org/10.1038/mtna.2013.49
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