In vivo site-specific engineering to reprogram T cells | Nature

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Subjects

- Cancer immunotherapy

- Genetic engineering

- Immunotherapy

- Tissue engineering

Abstract

Engineered T cells, reprogrammed to express chimeric antigen receptors (CAR) or T cell receptors (TCR), have transformed cancer treatment and are being explored as therapeutics for autoimmune and infectious diseases. Enhancing T cell function through genome editing, either by disrupting endogenous genes or precisely inserting DNA payloads, has shown considerable promise1. However, the ex vivo manufacturing process is lengthy and costly, limiting accessibility of these therapies. In vivo generation of CAR T cells could overcome these barriers, but current methods rely either on transient expression with limited durability, or on random integration of DNA payloads that lack specificity. Here we demonstrate that stable and cell-specific transgene expression can be achieved through in vivo site-specific integration of large DNA payloads. We developed a two-vector system to deliver CRISPR–Cas9 ribonucleoproteins and a DNA donor template, using enveloped delivery vehicles and adeno-associated viruses, respectively. We optimized both vectors for T cell-specific delivery and gene-targeting efficiency. By integrating a CAR transgene into a T cell-specific locus, we generate therapeutic levels of CAR T cells in vivo in humanized mouse models of B cell aplasia, and haematological and solid malignancies. These findings offer a pathway to more efficient, precise and widely accessible T cell therapies.

Main

CAR T cells are a promising treatment for haematological malignancies; to date, there are seven CAR T cell therapies that have been approved by the US Food and Drug Administration. Standard-of-care CAR T cell therapy requires patient-specific manufacturing, limited by variable product quality, long production times and high cost. CARs are usually delivered using retroviral vectors, producing heterogeneous expression from random integration2,3. Using CRISPR–Cas9 and adeno-associated virus (AAV)-mediated homology-directed repair (HDR), we targeted CAR integration into the endogenous human TCR alpha locus (TRAC). TRAC-CAR T cells display dynamic CAR expression that delays exhaustion and improves tumour control in xenograft and immunocompetent models1,4. This work has been critical for the development of allogeneic CAR T cell therapy, as it disrupts the TCR after transgene insertion—a necessary step to limit graft-versus-host disease (GvHD)5. Clinical trials using allogeneic TRAC-CAR T cells generated from healthy donors or induced pluripotent stem cells have achieved complete responses in patients with haematological malignancies when associated with deep lymphodepleting preconditioning6. Allogeneic approaches could address manufacturing limitations by creating off-the-shelf products from healthy donors. However, allogeneic CAR T cells are eventually rejected and frequent relapses have been observed7.

Direct in vivo CAR T cell generation may circumvent hurdles associated with leukapheresis and manufacturing. It might also promote the formation of a less differentiated CAR T cell pool8, a feature associated with improved antitumour activity9,10,11. Thus far, efforts to generate CAR T cells in vivo have used randomly integrating viral vectors for constitutive CAR expression or lipid nanoparticles (LNPs) that result in transient CAR expression12,13,14,15,16. Both modalities have been recently validated in non-human primates17,18 and, more recently, evaluated in a phase I trial19. These methods face challenges, including efficient gene delivery to therapeutic doses and risks of off-target transduction. Both delivery and CAR expression should be T cell specific, as off-target engineering of haematopoietic stem cells (HSCs) could lead to transformational mutagenesis20, and CAR expression in tumour cells could prevent cell surface expression of the CAR target and cause antigen-negative relapse21. LNP delivery of CAR mRNA leads to transient CAR expression, which prevents the risk of insertional mutagenesis or stable tumour cell expression, but required dosing remains unclear. Lentiviral vector envelopes can be engineered to provide improved specificity towards T cells12,16,22,23,24, but any transduced non-T cells would also express the CAR unless a lineage-specific promoter is used and, while rare, are at potential risk of insertional mutagenesis25,26. We hypothesized that integrating a promoterless CAR transgene at the TRAC locus in vivo would achieve T cell-specific and physiological CAR expression, while bypassing ex vivo cell manufacturing. To date, site-specific integration of large DNA payloads in human T cells in vivo remains elusive.

Here we develop a method combining AAVs with enveloped delivery vehicles (EDVs) to perform site-specific transgene integration in primary human T cells in vivo. By optimizing both the AAV and EDV tools for improved cell-specific delivery and resistance to human neutrali