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Transduction of modified factor VIII gene improves lentiviral gene therapy efficacy for hemophilia A
Hemophilia A (HA) is a bleeding disorder caused by deficiency of the coagulation factor VIII (F8). F8 replacement is standard of care, whereas gene therapy (F8 gene) for HA is an attractive investigational approach. However, the large size of the F8 gene and the immunogenicity of the product present...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Society for Biochemistry and Molecular Biology
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8649223/ https://www.ncbi.nlm.nih.gov/pubmed/34774524 http://dx.doi.org/10.1016/j.jbc.2021.101397 |
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author | Gong, Jie Chung, Tsai-Hua Zheng, Jie Zheng, Huyong Chang, Lung-Ji |
author_facet | Gong, Jie Chung, Tsai-Hua Zheng, Jie Zheng, Huyong Chang, Lung-Ji |
author_sort | Gong, Jie |
collection | PubMed |
description | Hemophilia A (HA) is a bleeding disorder caused by deficiency of the coagulation factor VIII (F8). F8 replacement is standard of care, whereas gene therapy (F8 gene) for HA is an attractive investigational approach. However, the large size of the F8 gene and the immunogenicity of the product present challenges in development of the F8 gene therapy. To resolve these problems, we synthesized a shortened F8 gene (F8-BDD) and cloned it into a lentiviral vector (LV). The F8-BDD produced mainly short cleaved inactive products in LV-transduced cells. To improve F8 functionality, we designed two novel F8-BDD genes, one with an insertion of eight specific N-glycosylation sites (F8-N8) and another which restored all N-glycosylation sites (F8-299) in the B domain. Although the overall protein expression was reduced, high coagulation activity (>100-fold) was detected in the supernatants of LV-F8-N8- and LV-F8-299-transduced cells. Protein analysis of F8 and the procoagulation cofactor, von Willebrand Factor, showed enhanced interaction after restoration of B domain glycosylation using F8-299. HA mouse hematopoietic stem cell transplantation studies illustrated that the bleeding phenotype was corrected after LV-F8-N8 or -299 gene transfer into the hematopoietic stem cells. Importantly, the F8-299 modification markedly reduced immunogenicity of the F8 protein in these HA mice. In conclusion, the modified F8-299 gene could be efficiently packaged into LV and, although with reduced expression, produced highly stable and functional F8 protein that corrected the bleeding phenotype without inhibitory immunogenicity. We anticipate that these results will be beneficial in the development of gene therapies against HA. |
format | Online Article Text |
id | pubmed-8649223 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | American Society for Biochemistry and Molecular Biology |
record_format | MEDLINE/PubMed |
spelling | pubmed-86492232021-12-17 Transduction of modified factor VIII gene improves lentiviral gene therapy efficacy for hemophilia A Gong, Jie Chung, Tsai-Hua Zheng, Jie Zheng, Huyong Chang, Lung-Ji J Biol Chem Research Article Hemophilia A (HA) is a bleeding disorder caused by deficiency of the coagulation factor VIII (F8). F8 replacement is standard of care, whereas gene therapy (F8 gene) for HA is an attractive investigational approach. However, the large size of the F8 gene and the immunogenicity of the product present challenges in development of the F8 gene therapy. To resolve these problems, we synthesized a shortened F8 gene (F8-BDD) and cloned it into a lentiviral vector (LV). The F8-BDD produced mainly short cleaved inactive products in LV-transduced cells. To improve F8 functionality, we designed two novel F8-BDD genes, one with an insertion of eight specific N-glycosylation sites (F8-N8) and another which restored all N-glycosylation sites (F8-299) in the B domain. Although the overall protein expression was reduced, high coagulation activity (>100-fold) was detected in the supernatants of LV-F8-N8- and LV-F8-299-transduced cells. Protein analysis of F8 and the procoagulation cofactor, von Willebrand Factor, showed enhanced interaction after restoration of B domain glycosylation using F8-299. HA mouse hematopoietic stem cell transplantation studies illustrated that the bleeding phenotype was corrected after LV-F8-N8 or -299 gene transfer into the hematopoietic stem cells. Importantly, the F8-299 modification markedly reduced immunogenicity of the F8 protein in these HA mice. In conclusion, the modified F8-299 gene could be efficiently packaged into LV and, although with reduced expression, produced highly stable and functional F8 protein that corrected the bleeding phenotype without inhibitory immunogenicity. We anticipate that these results will be beneficial in the development of gene therapies against HA. American Society for Biochemistry and Molecular Biology 2021-11-10 /pmc/articles/PMC8649223/ /pubmed/34774524 http://dx.doi.org/10.1016/j.jbc.2021.101397 Text en © 2021 The Authors https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). |
spellingShingle | Research Article Gong, Jie Chung, Tsai-Hua Zheng, Jie Zheng, Huyong Chang, Lung-Ji Transduction of modified factor VIII gene improves lentiviral gene therapy efficacy for hemophilia A |
title | Transduction of modified factor VIII gene improves lentiviral gene therapy efficacy for hemophilia A |
title_full | Transduction of modified factor VIII gene improves lentiviral gene therapy efficacy for hemophilia A |
title_fullStr | Transduction of modified factor VIII gene improves lentiviral gene therapy efficacy for hemophilia A |
title_full_unstemmed | Transduction of modified factor VIII gene improves lentiviral gene therapy efficacy for hemophilia A |
title_short | Transduction of modified factor VIII gene improves lentiviral gene therapy efficacy for hemophilia A |
title_sort | transduction of modified factor viii gene improves lentiviral gene therapy efficacy for hemophilia a |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8649223/ https://www.ncbi.nlm.nih.gov/pubmed/34774524 http://dx.doi.org/10.1016/j.jbc.2021.101397 |
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