Generating universal anti-CD19 CAR T cells with a defined memory phenotype by CRISPR/Cas9 editing and safety evaluation of the transcriptome.

Introduction: Chimeric antigen receptor-expressing T cells (CAR T cells) have revolutionized cancer treatment, particularly in B cell malignancies. However, the use of autologous T cells for CAR T therapy presents several limitations, including high costs, variable efficacy, and adverse effects linked to cell phenotype. Methods: To overcome these challenges, we developed a strategy to generate universal and safe anti-CD19 CAR T cells with a defined memory phenotype. Our approach utilizes CRISPR/Cas9 technology to target and eliminate the B2M and TRAC genes, reducing graft-versus-host and host-versus-graft responses. Additionally, we selected less differentiated T cells to improve the stability and persistence of the universal CAR T cells. The safety of this method was assessed using our CRISPRroots transcriptome analysis pipeline, which ensures successful gene knockout and the absence of unintended off-target effects on gene expression or transcriptome sequence. Results: In vitro experiments demonstrated the successful generation of functional universal CAR T cells. These cells exhibited potent lytic activity against tumor cells and a reduced cytokine secretion profile. The CRISPRroots analysis confirmed effective gene knockout and no unintended off-target effects, validating it as a pioneering tool for on/off-target and transcriptome analysis in genome editing experiments. Discussion: Our findings establish a robust pipeline for manufacturing safe, universal CAR T cells with a favorable memory phenotype. This approach has the potential to address the current limitations of autologous CAR T cell therapy, offering a more stable and persistent treatment option with reduced adverse effects. The use of CRISPRroots enhances the reliability and safety of gene editing in the development of CAR T cell therapies. Conclusion: We have developed a potent and reliable method for producing universal CAR T cells with a defined memory phenotype, demonstrating both efficacy and safety in vitro. This innovative approach could significantly improve the therapeutic landscape for patients with B cell malignancies.

Lentiviral vectors for inducible, transactivator-free advanced therapy medicinal products: Application to CAR-T cells.

Controlling transgene expression through an externally administered inductor is envisioned as a potent strategy to improve safety and efficacy of gene therapy approaches. Generally, inducible ON systems require a chimeric transcription factor (transactivator) that becomes activated by an inductor, which is not optimal for clinical translation due to their toxicity. We generated previously the first all-in-one, transactivator-free, doxycycline (Dox)-responsive (Lent-On-Plus or LOP) lentiviral vectors (LVs) able to control transgene expression in human stem cells. Here, we have generated new versions of the LOP LVs and have analyzed their applicability for the generation of inducible advanced therapy medicinal products (ATMPs) with special focus on primary human T cells. We have shown that, contrary to all other cell types analyzed, an Is2 insulator must be inserted into the 3' long terminal repeat of the LOP LVs in order to control transgene expression in human primary T cells. Importantly, inducible primary T cells generated by the LOPIs2 LVs are responsive to ultralow doses of Dox and have no changes in phenotype or function compared with untransduced T cells. We validated the LOPIs2 system by generating inducible CAR-T cells that selectively kill CD19+ cells in the presence of Dox. In summary, we describe here the first transactivator-free, all-one-one system capable of generating Dox-inducible ATMPs.

Efficacy and safety of universal (TCRKO) ARI-0001 CAR-T cells for the treatment of B-cell lymphoma.

Autologous T cells expressing the Chimeric Antigen Receptor (CAR) have been approved as advanced therapy medicinal products (ATMPs) against several hematological malignancies. However, the generation of patient-specific CAR-T products delays treatment and precludes standardization. Allogeneic off-the-shelf CAR-T cells are an alternative to simplify this complex and time-consuming process. Here we investigated safety and efficacy of knocking out the TCR molecule in ARI-0001 CAR-T cells, a second generation αCD19 CAR approved by the Spanish Agency of Medicines and Medical Devices (AEMPS) under the Hospital Exemption for treatment of patients older than 25 years with Relapsed/Refractory acute B cell lymphoblastic leukemia (B-ALL). We first analyzed the efficacy and safety issues that arise during disruption of the TCR gene using CRISPR/Cas9. We have shown that edition of TRAC locus in T cells using CRISPR as ribonuleorproteins allows a highly efficient TCR disruption (over 80%) without significant alterations on T cells phenotype and with an increased percentage of energetic mitochondria. However, we also found that efficient TCRKO can lead to on-target large and medium size deletions, indicating a potential safety risk of this procedure that needs monitoring. Importantly, TCR edition of ARI-0001 efficiently prevented allogeneic responses and did not detectably alter their phenotype, while maintaining a similar anti-tumor activity ex vivo and in vivo compared to unedited ARI-0001 CAR-T cells. In summary, we showed here that, although there are still some risks of genotoxicity due to genome editing, disruption of the TCR is a feasible strategy for the generation of functional allogeneic ARI-0001 CAR-T cells. We propose to further validate this protocol for the treatment of patients that do not fit the requirements for standard autologous CAR-T cells administration.

Erratum: Physiological lentiviral vectors for the generation of improved CAR-T cells.

[This corrects the article DOI: 10.1016/j.omto.2022.05.003.].

Physiological lentiviral vectors for the generation of improved CAR-T cells.

Anti-CD19 chimeric antigen receptor (CAR)-T cells have achieved impressive outcomes for the treatment of relapsed and refractory B-lineage neoplasms. However, important limitations still remain due to severe adverse events (i.e., cytokine release syndrome and neuroinflammation) and relapse of 40%-50% of the treated patients. Most CAR-T cells are generated using retroviral vectors with strong promoters that lead to high CAR expression levels, tonic signaling, premature exhaustion, and overstimulation, reducing efficacy and increasing side effects. Here, we show that lentiviral vectors (LVs) expressing the transgene through a WAS gene promoter (AW-LVs) closely mimic the T cell receptor (TCR)/CD3 expression kinetic upon stimulation. These AW-LVs can generate improved CAR-T cells as a consequence of their moderate and TCR-like expression profile. Compared with CAR-T cells generated with human elongation factor α (EF1α)-driven-LVs, AW-CAR-T cells exhibited lower tonic signaling, higher proportion of naive and stem cell memory T cells, less exhausted phenotype, and milder secretion of tumor necrosis factor alpha (TNF-α) and interferon (IFN)-É£ after efficient destruction of CD19+ lymphoma cells, both in vitro and in vivo. Moreover, we also showed their improved efficiency using an in vitro CD19+ pancreatic tumor model. We finally demonstrated the feasibility of large-scale manufacturing of AW-CAR-T cells in guanosine monophosphate (GMP)-like conditions. Based on these data, we propose the use of AW-LVs for the generation of improved CAR-T products.

Externally-Controlled Systems for Immunotherapy: From Bench to Bedside.

Immunotherapy is a very promising therapeutic approach against cancer that is particularly effective when combined with gene therapy. Immuno-gene therapy approaches have led to the approval of four advanced therapy medicinal products (ATMPs) for the treatment of p53-deficient tumors (Gendicine and Imlygic), refractory acute lymphoblastic leukemia (Kymriah) and large B-cell lymphomas (Yescarta). In spite of these remarkable successes, immunotherapy is still associated with severe side effects for CD19+ malignancies and is inefficient for solid tumors. Controlling transgene expression through an externally administered inductor is envisioned as a potent strategy to improve safety and efficacy of immunotherapy. The aim is to develop smart immunogene therapy-based-ATMPs, which can be controlled by the addition of innocuous drugs or agents, allowing the clinicians to manage the intensity and durability of the therapy. In the present manuscript, we will review the different inducible, versatile and externally controlled gene delivery systems that have been developed and their applications to the field of immunotherapy. We will highlight the advantages and disadvantages of each system and their potential applications in clinics.

Using Gene Editing Approaches to Fine-Tune the Immune System.

Genome editing technologies not only provide unprecedented opportunities to study basic cellular system functionality but also improve the outcomes of several clinical applications. In this review, we analyze various gene editing techniques used to fine-tune immune systems from a basic research and clinical perspective. We discuss recent advances in the development of programmable nucleases, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas-associated nucleases. We also discuss the use of programmable nucleases and their derivative reagents such as base editing tools to engineer immune cells via gene disruption, insertion, and rewriting of T cells and other immune components, such natural killers (NKs) and hematopoietic stem and progenitor cells (HSPCs). In addition, with regard to chimeric antigen receptors (CARs), we describe how different gene editing tools enable healthy donor cells to be used in CAR T therapy instead of autologous cells without risking graft-versus-host disease or rejection, leading to reduced adoptive cell therapy costs and instant treatment availability for patients. We pay particular attention to the delivery of therapeutic transgenes, such as CARs, to endogenous loci which prevents collateral damage and increases therapeutic effectiveness. Finally, we review creative innovations, including immune system repurposing, that facilitate safe and efficient genome surgery within the framework of clinical cancer immunotherapies.