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

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

Molecular Therapy: Methods & Clinical Development (Sep 2020)

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

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

Affiliations

Dahae Hailey Bae: Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, US Food and Drug Administration (FDA), Silver Spring, MD, USA
Michael Marino: Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, US Food and Drug Administration (FDA), Silver Spring, MD, USA
Brian Iaffaldano: Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, US Food and Drug Administration (FDA), Silver Spring, MD, USA
Sydney Fenstermaker: Office of Biostatistics and Epidemiology, Center for Biologics Evaluation and Research, FDA, Silver Spring, MD, USA
Sandra Afione: AAV Biology Section, National Institute of Dental and Craniofacial Research, NIH, Bethesda, MD, USA
Takele Argaw: Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, US Food and Drug Administration (FDA), Silver Spring, MD, USA
Jacob McCright: Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, US Food and Drug Administration (FDA), Silver Spring, MD, USA
Anna Kwilas: Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, US Food and Drug Administration (FDA), Silver Spring, MD, USA
John A. Chiorini: AAV Biology Section, National Institute of Dental and Craniofacial Research, NIH, Bethesda, MD, USA
Andrew E. Timmons: Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, US Food and Drug Administration (FDA), Silver Spring, MD, USA
Jakob Reiser: Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, US Food and Drug Administration (FDA), Silver Spring, MD, USA; Corresponding author: Jakob Reiser, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, US Food and Drug Administration (FDA), Silver Spring, MD, USA.

Journal volume & issue: Vol. 18
pp. 631 – 638

Abstract

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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.

Published in Molecular Therapy: Methods & Clinical Development

ISSN: 2329-0501 (Online)
Publisher: Elsevier
Country of publisher: United States
LCC subjects: Science: Biology (General): Genetics; Science: Biology (General): Cytology
Website: http://www.cell.com/molecular-therapy-family/methods/latest-content

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