Novel extragenic genomic safe harbors for precise therapeutic T-cell engineering

• Published in: Blood 2023-06, Vol.141 (22), p.2698-2712
• Main Authors: Odak, Ashlesha, Yuan, Han, Feucht, Judith, Cantu, Vito Adrian, Mansilla-Soto, Jorge, Kogel, Friederike, Eyquem, Justin, Everett, John, Bushman, Frederic D., Leslie, Christina S., Sadelain, Michel
• Format: Article
• Language: English
• Subjects: Animals; Antigens, CD19; Cell Engineering; Genetic Vectors; Genomics; Humans; Immunotherapy, Adoptive; Mice; Receptors, Antigen, T-Cell; T-Lymphocytes
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.2022018924

•CAR T-cell engineering at GSH6 achieves long-term tumor control.•Validated criteria identify targetable extragenic GSHs. [Display omitted] Cell therapies that rely on engineered immune cells can be enhanced by achieving uniform and controlled transgene expression in order to maximize T-cell function and achieve predictable patient responses. Although they are effective, current genetic engineering strategies that use γ-retroviral, lentiviral, and transposon-based vectors to integrate transgenes, unavoidably produce variegated transgene expression in addition to posing a risk of insertional mutagenesis. In the setting of chimeric antigen receptor (CAR) therapy, inconsistent and random CAR expression may result in tonic signaling, T-cell exhaustion, and variable T-cell persistence. Here, we report and validate an algorithm for the identification of extragenic genomic safe harbors (GSH) that can be efficiently targeted for DNA integration and can support sustained and predictable CAR expression in human peripheral blood T cells. The algorithm is based on 7 criteria established to minimize genotoxicity by directing transgene integration away from functionally important genomic elements, maximize efficient CRISPR/Cas9–mediated targeting, and avert transgene silencing over time. T cells engineered to express a CD19 CAR at GSH6, which meets all 7 criteria, are curative at low cell dose in a mouse model of acute lymphoblastic leukemia, matching the potency of CAR T cells engineered at the TRAC locus and effectively resisting tumor rechallenge 100 days after their infusion. The identification of functional extragenic GSHs thus expands the human genome available for therapeutic precision engineering. Targeted integration of chimeric antigen receptor (CAR) transgenes in defined genetic locations is anticipated to improve sustained expression and reduce risks associated with conventional gene transfer methods, such as insertional mutagenesis. Using a CRISPR-Cas9–assisted knock-in technique, Odak et al investigated locations in the noncoding genome that provide stable expression of CAR transgenes in human T cells, identifying GSH6 as such a site that can achieve long-term tumor control. The authors’ data expand our ability to engineer potent CAR T-cell products and provide resources for the identification of other extragenic genomic safe harbor locations.