Design and Testing of Vector-Producing HEK293T Cells Bearing a Genomic Deletion of the SV40 T Antigen Coding Region

Design and Testing of Vector-Producing HEK293T Cells Bearing a Genomic Deletion of the SV40 T Antigen Coding Region

The use of the human embryonic kidney (HEK) 293T cell line to manufacture vectors for in vivo applications raises safety concerns due to the presence of SV40 T antigen-encoding sequences. We used CRISPR-Cas9 genome editing to remove the SV40 T antigen-encoding sequences from HEK293T cells by transfe...

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Bibliographic Details
Main Authors: Dahae Hailey Bae, Michael Marino, Brian Iaffaldano, Sydney Fenstermaker, Sandra Afione, Takele Argaw, Jacob McCright, Anna Kwilas, John A. Chiorini, Andrew E. Timmons, Jakob Reiser
Format: Article
Language:English
Published: Elsevier 2020-09-01
Series:Molecular Therapy: Methods & Clinical Development
Subjects:
Lentivirus
AAV
vector manufacturing
HEK293T cells
SV40 T antigen
CRISPR/Cas9
Online Access:http://www.sciencedirect.com/science/article/pii/S2329050120301558
Description
Summary:The use of the human embryonic kidney (HEK) 293T cell line to manufacture vectors for in vivo applications raises safety concerns due to the presence of SV40 T antigen-encoding sequences. We used CRISPR-Cas9 genome editing to remove the SV40 T antigen-encoding sequences from HEK293T cells by transfecting them with a recombinant plasmid expressing Cas9 and two distinct single guide RNAs (sgRNAs) corresponding to the beginning and end of the T antigen coding region. Cell clones lacking T antigen-encoding sequences were identified using PCR. Whole-genome (WG) and targeted locus amplification (TLA) sequencing of the parental HEK293T cell line revealed multiple SV40 T antigen-encoding sequences replacing cellular sequences on chromosome 3. The putative T antigen null clones demonstrated a loss of sequence reads mapping to T antigen-encoding sequences. Western blot analysis of cell extracts prepared from the T antigen null clones confirmed that the SV40 large and small T antigen proteins were absent. Lentiviral vectors produced using the T antigen null clones exhibited titers up to 1.5 × 107 transducing units (TU)/mL, while the titers obtained from the parent HEK293T cell line were up to 4 × 107 TU/mL. The capacity of the T antigen-negative cells to produce high titer adeno-associated virus (AAV) vectors was also evaluated. The results obtained revealed that the lack of T antigen sequences did not impact AAV vector titers.
ISSN:2329-0501

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