Abstract
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.
Highlights
Lentiviral vectors are typically produced using the human embryonic kidney (HEK) 293T cell line
Despite the absence of the SV40 T antigen, we found that T antigen knockout clones retained the high-titer phenotype of HEK293T cells for lentiviral vector production
Assessing Lentiviral Vector Titers Obtained Using HEK293 and HEK293T Cell Clones To determine the influence of clonal variation on lentiviral vector titers, clonal populations of HEK293 and HEK293T cells were established by limiting dilution
Summary
Lentiviral vectors are typically produced using the human embryonic kidney (HEK) 293T cell line. Compared to the parental HEK293 cell line, lentiviral vector titers obtained using HEK293T cells are higher.[1,2] The original HEK293 cell line was derived by transformation of a human primary embryonic kidney cell line using mechanically sheared DNA of adenovirus type 5 (Ad5).[3] A 4-kb Ad5 DNA fragment encoding the E1A/E1B proteins was later shown to have integrated into chromosome 19, resulting in transformation.[4]. Next-generation sequencing (NGS) of SV40 T antigen-negative cell clones, as well as the parental HEK293T cell line, confirmed the presence of deletions within the SV40 large T antigen coding sequence. Targeted locus amplification (TLA) sequencing[9,10] allowed further characterization of the integration site of the pRTAK SV40 T antigen plasmid originally used to derive the HEK293T cell line.[11] This analysis revealed multiple plasmid copies within a 550-kb deletion of cellular sequences on chromosome 3
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