High efficiency closed-system gene transfer using automated spinoculation

Abstract Background Gene transfer is an important tool for cellular therapies. Lentiviral vectors are most effectively transferred into lymphocytes or hematopoietic progenitor cells using spinoculation. To enable cGMP (current Good Manufacturing Practice)-compliant cell therapy production, we develo...

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Autores principales:	Victoria Ann Remley, Jianjian Jin, Sarmila Sarkar, Larry Moses, Michaela Prochazkova, Yihua Cai, Lipei Shao, Hui Liu, Tatyana Fuksenko, Ping Jin, David F. Stroncek, Steven L. Highfill
Formato:	article
Lenguaje:	EN
Publicado:	BMC 2021
Materias:	Sepax Spinoculation CAR T-cell Gene transfer Medicine R
Acceso en línea:	https://doaj.org/article/7a27ea1b72e34fa48e8d324754b08462
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spelling	oai:doaj.org-article:7a27ea1b72e34fa48e8d324754b084622021-11-28T12:06:47ZHigh efficiency closed-system gene transfer using automated spinoculation10.1186/s12967-021-03126-41479-5876https://doaj.org/article/7a27ea1b72e34fa48e8d324754b084622021-11-01T00:00:00Zhttps://doi.org/10.1186/s12967-021-03126-4https://doaj.org/toc/1479-5876Abstract Background Gene transfer is an important tool for cellular therapies. Lentiviral vectors are most effectively transferred into lymphocytes or hematopoietic progenitor cells using spinoculation. To enable cGMP (current Good Manufacturing Practice)-compliant cell therapy production, we developed and compared a closed-system spinoculation method that uses cell culture bags, and an automated closed system spinoculation method to decrease technician hands on time and reduce the likelihood for microbial contamination. Methods Sepax spinoculation, bag spinoculation, and static bag transduction without spinoculation were compared for lentiviral gene transfer in lymphocytes collected by apheresis. The lymphocytes were transduced once and cultured for 9 days. The lentiviral vectors tested encoded a CD19/CD22 Bispecific Chimeric Antigen Receptor (CAR), a FGFR4-CAR, or a CD22-CAR. Sepax spinoculation times were evaluated by testing against bag spinoculation and static transduction to optimize the Sepax spin time. The Sepax spinoculation was then used to test the transduction of different CAR vectors. The performance of the process using healthy donor and a patient sample was evaluated. Functional assessment was performed of the CD19/22 and CD22 CAR T-cells using killing assays against the NALM6 tumor cell line and cytokine secretion analysis. Finally, gene expression of the transduced T-cells was examined to determine if there were any major changes that may have occurred as a result of the spinoculation process. Results The process of spinoculation lead to significant enhancement in gene transfer. Sepax spinoculation using a 1-h spin time showed comparable transduction efficiency to the bag spinoculation, and much greater than the static bag transduction method (83.4%, 72.8%, 35.7% n = 3). The performance of three different methods were consistent for all lentiviral vectors tested and no significant difference was observed when using starting cells from healthy donor versus a patient sample. Sepax spinoculation does not affect the function of the CAR T-cells against tumor cells, as these cells appeared to kill target cells equally well. Spinoculation also does not appear to affect gene expression patterns that are necessary for imparting function on the cell. Conclusions Closed system-bag spinoculation resulted in more efficient lymphocyte gene transfer than standard bag transductions without spinoculation. This method is effective for both retroviral and lentiviral vector gene transfer in lymphocytes and may be a feasible approach for gene transfer into other cell types including hematopoietic and myeloid progenitors. Sepax spinoculation further improved upon the process by offering an automated, closed system approach that significantly decreased hands-on time while also decreasing the risk of culture bag tears and microbial contamination.Victoria Ann RemleyJianjian JinSarmila SarkarLarry MosesMichaela ProchazkovaYihua CaiLipei ShaoHui LiuTatyana FuksenkoPing JinDavid F. StroncekSteven L. HighfillBMCarticleSepaxSpinoculationCAR T-cellGene transferMedicineRENJournal of Translational Medicine, Vol 19, Iss 1, Pp 1-15 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Sepax Spinoculation CAR T-cell Gene transfer Medicine R
spellingShingle	Sepax Spinoculation CAR T-cell Gene transfer Medicine R Victoria Ann Remley Jianjian Jin Sarmila Sarkar Larry Moses Michaela Prochazkova Yihua Cai Lipei Shao Hui Liu Tatyana Fuksenko Ping Jin David F. Stroncek Steven L. Highfill High efficiency closed-system gene transfer using automated spinoculation
description	Abstract Background Gene transfer is an important tool for cellular therapies. Lentiviral vectors are most effectively transferred into lymphocytes or hematopoietic progenitor cells using spinoculation. To enable cGMP (current Good Manufacturing Practice)-compliant cell therapy production, we developed and compared a closed-system spinoculation method that uses cell culture bags, and an automated closed system spinoculation method to decrease technician hands on time and reduce the likelihood for microbial contamination. Methods Sepax spinoculation, bag spinoculation, and static bag transduction without spinoculation were compared for lentiviral gene transfer in lymphocytes collected by apheresis. The lymphocytes were transduced once and cultured for 9 days. The lentiviral vectors tested encoded a CD19/CD22 Bispecific Chimeric Antigen Receptor (CAR), a FGFR4-CAR, or a CD22-CAR. Sepax spinoculation times were evaluated by testing against bag spinoculation and static transduction to optimize the Sepax spin time. The Sepax spinoculation was then used to test the transduction of different CAR vectors. The performance of the process using healthy donor and a patient sample was evaluated. Functional assessment was performed of the CD19/22 and CD22 CAR T-cells using killing assays against the NALM6 tumor cell line and cytokine secretion analysis. Finally, gene expression of the transduced T-cells was examined to determine if there were any major changes that may have occurred as a result of the spinoculation process. Results The process of spinoculation lead to significant enhancement in gene transfer. Sepax spinoculation using a 1-h spin time showed comparable transduction efficiency to the bag spinoculation, and much greater than the static bag transduction method (83.4%, 72.8%, 35.7% n = 3). The performance of three different methods were consistent for all lentiviral vectors tested and no significant difference was observed when using starting cells from healthy donor versus a patient sample. Sepax spinoculation does not affect the function of the CAR T-cells against tumor cells, as these cells appeared to kill target cells equally well. Spinoculation also does not appear to affect gene expression patterns that are necessary for imparting function on the cell. Conclusions Closed system-bag spinoculation resulted in more efficient lymphocyte gene transfer than standard bag transductions without spinoculation. This method is effective for both retroviral and lentiviral vector gene transfer in lymphocytes and may be a feasible approach for gene transfer into other cell types including hematopoietic and myeloid progenitors. Sepax spinoculation further improved upon the process by offering an automated, closed system approach that significantly decreased hands-on time while also decreasing the risk of culture bag tears and microbial contamination.
format	article
author	Victoria Ann Remley Jianjian Jin Sarmila Sarkar Larry Moses Michaela Prochazkova Yihua Cai Lipei Shao Hui Liu Tatyana Fuksenko Ping Jin David F. Stroncek Steven L. Highfill
author_facet	Victoria Ann Remley Jianjian Jin Sarmila Sarkar Larry Moses Michaela Prochazkova Yihua Cai Lipei Shao Hui Liu Tatyana Fuksenko Ping Jin David F. Stroncek Steven L. Highfill
author_sort	Victoria Ann Remley
title	High efficiency closed-system gene transfer using automated spinoculation
title_short	High efficiency closed-system gene transfer using automated spinoculation
title_full	High efficiency closed-system gene transfer using automated spinoculation
title_fullStr	High efficiency closed-system gene transfer using automated spinoculation
title_full_unstemmed	High efficiency closed-system gene transfer using automated spinoculation
title_sort	high efficiency closed-system gene transfer using automated spinoculation
publisher	BMC
publishDate	2021
url	https://doaj.org/article/7a27ea1b72e34fa48e8d324754b08462
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High efficiency closed-system gene transfer using automated spinoculation

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