improving-crispr-cas9-screens-car-t-cells-refined-method-for-library

Improving CRISPR-Cas9 Screens in CAR T Cells: A Refined Method for Library Preparation

Chimeric antigen receptor (CAR) T cell therapies have demonstrated remarkable efficacy in several hematological malignancies, yet their success has not ...

Cima Universidad de Navarra, IdiSNA

Primary Hyperoxaluria Type I (PH1) is a rare autosomal recessive metabolic disorder characterized by defects in enzymes involved in glyoxylate metabolism. PH1 is a life-threatening disease caused by the absence, deficiency or mistargeting of the hepatic alanine-glyoxylate aminotransferase (AGT) enzyme. A human induced pluripotent stem cell (iPSC) line was generated from dermal fibroblasts of a PH1 patient being compound heterozygous for the most common mutation c.508G>A (G170R), a mistargeting mutation, and c.364C>T (R122*), a previously reported nonsense mutation in AGTX. This iPSC line offers a useful resource to study the disease pathophysiology and a cell-based model for drug development.

Generation of an induced pluripotent stem cell line (CIMAi001-A) from a compound heterozygous Primary Hyperoxaluria Type I (PH1) patient carrying p.G170R and p.R122* mutations in the AGXT gene.

Genome-editing strategies, especially CRISPR-Cas9 systems, have substantially increased the efficiency of innovative therapeutic approaches for monogenic diseases such as primary hyperoxalurias (PHs). We have previously demonstrated that inhibition of glycolate oxidase using CRISPR-Cas9 systems represents a promising therapeutic option for PH type I (PH1). Here, we extended our work evaluating the efficacy of liver-specific inhibition of lactate dehydrogenase (LDH), a key enzyme responsible for converting glyoxylate to oxalate; this strategy would not be limited to PH1, being applicable to other PH subtypes. In this work, we demonstrate a liver-specific inhibition of LDH that resulted in a drastic reduction of LDH levels in the liver of PH1 and PH3 mice after a single-dose delivery of AAV8 vectors expressing the CRISPR-Cas9 system, resulting in reduced urine oxalate levels and kidney damage without signs of toxicity. Deep sequencing analysis revealed that this approach was safe and specific, with no off-targets detected in the liver of treated animals and no on-target/off-tissue events. Altogether, our data provide evidence that  genome editing using CRISPR-Cas9 systems would represent a valuable tool for improved therapeutic approaches for PH.

 CRISPR-Cas9 inhibition of hepatic LDH as treatment of primary hyperoxaluria.

Identification of new markers associated with long-term efficacy in patients treated with CAR T cells is a current medical need, particularly in diseases such as multiple myeloma. In this study, we address the impact of CAR density on the functionality of BCMA CAR T cells. Functional and transcriptional studies demonstrate that CAR T cells with high expression of the CAR construct show an increased tonic signaling with up-regulation of exhaustion markers and increased in vitro cytotoxicity but a decrease in in vivo BM infiltration. Characterization of gene regulatory networks using scRNA-seq identified regulons associated to activation and exhaustion up-regulated in CAR T cells, providing mechanistic insights behind differential functionality of these cells. Last, we demonstrate that patients treated with CAR T cell products enriched in CAR T cells show a significantly worse clinical response in several hematological malignancies. In summary, our work demonstrates that CAR density plays an important role in CAR T activity with notable impact on clinical response.

CAR density influences antitumoral efficacy of BCMA CAR T cells and correlates with clinical outcome.

Despite the potential of CAR-T therapies for hematological malignancies, their efficacy in patients with relapse and refractory Acute Myeloid Leukemia has been limited. The aim of our study has been to develop and manufacture a CAR-T cell product that addresses some of the current limitations. We initially compared the phenotype of T cells from AML patients and healthy young and elderly controls. This analysis showed that T cells from AML patients displayed a predominantly effector phenotype, with increased expression of activation (CD69 and HLA-DR) and exhaustion markers (PD1 and LAG3), in contrast to the enriched memory phenotype observed in healthy donors. This differentiated and more exhausted phenotype was also observed, and corroborated by transcriptomic analyses, in CAR-T cells from AML patients engineered with an optimized CAR construct targeting CD33, resulting in a decreased  antitumoral efficacy evaluated in xenograft AML models. To overcome some of these limitations we have combined CRISPR-based genome editing technologies with virus-free gene-transfer strategies using  transposons, to generate CAR-T cells depleted of HLA-I and TCR complexes (HLA-I/TCR CAR-T cells) for allogeneic approaches. Our optimized protocol allows one-step generation of edited CAR-T cells that show a similar phenotypic profile to non-edited CAR-T cells, with equivalent  and  antitumoral efficacy. Moreover, genomic analysis of edited CAR-T cells revealed a safe integration profile of the vector, with no preferences for specific genomic regions, with highly specific editing of the HLA-I and TCR, without significant off-target sites. Finally, the production of edited CAR-T cells at a larger scale allowed the generation and selection of enough HLA-I/TCR CAR-T cells that would be compatible with clinical applications. In summary, our results demonstrate that CAR-T cells from AML patients, although functional, present phenotypic and functional features that could compromise their antitumoral efficacy, compared to CAR-T cells from healthy donors. The combination of CRISPR technologies with transposon-based delivery strategies allows the generation of HLA-I/TCR CAR-T cells, compatible with allogeneic approaches, that would represent a promising option for AML treatment.

Optimization of universal allogeneic CAR-T cells combining CRISPR and transposon-based technologies for treatment of acute myeloid leukemia.

Hematologic toxicity is a common side effect of chimeric antigen receptor T-cell (CAR-T) therapies, being particularly severe among patients with relapsed or refractory multiple myeloma (MM). In this study, we characterized 48 patients treated with B-cell maturation antigen (BCMA) CAR-T cells to understand kinetics of cytopenia, identify predictive factors, and determine potential mechanisms underlying these toxicities. We observed that overall incidence of cytopenia was 95.7%, and grade >3 thrombocytopenia and neutropenia, 1 month after infusion, was observed in 57% and 53% of the patients, respectively, being still present after 1 year in 4 and 3 patients, respectively. Baseline cytopenia and high peak inflammatory markers were highly correlated with cytopenia that persisted up to 3 months. To determine potential mechanisms underlying cytopenias, we evaluated the paracrine effect of BCMA CAR-T cells on hematopoietic stem and progenitor cell (HSPC) differentiation using an ex vivo myeloid differentiation model. Phenotypic analysis showed that supernatants from activated CAR-T cells (spCAR) halted HSPC differentiation, promoting more immature phenotypes, which could be prevented with a combination of interferon γ, tumor necrosis factor α/β, transforming growth factor β, interleukin-6 (IL-6) and IL-17 inhibitors. Single-cell RNA sequencing demonstrated upregulation of transcription factors associated with early stages of hematopoietic differentiation in the presence of spCAR (GATA2, RUNX1, CEBPA) and a decrease in the activity of key regulons involved in neutrophil and monocytic maturation (ID2 and MAFB). These results suggest that CAR-T activation induces HSPC maturation arrest through paracrine effects and provides potential treatments to mitigate the severity of this toxicity.

Molecular mechanisms promoting long-term cytopenia after BCMA CAR-T therapy in multiple myeloma.

Most CAR-T therapies rely on genetic T cell engineering with integrating viral vectors that, although effective, are associated with prohibitive costs. Here we have generated TranspoCART19 cells, a fully functional 4-1BB second-generation CAR-T cell product targeting CD19, fused to a truncated version of the human EGFR (hEGFRt) as reporter gene and safety switch, based on the  transposon delivery system. Our manufacturing protocol allowed generation of TranspoCART19 cells under GMP conditions, showing similar  and  antitumoral efficacy than conventional CAR-T cells generated with lentiviral vectors. Additionally, membrane expression of hEGFRt facilitated  CAR-T cell elimination after cetuximab administration. Safety analyses showed that TranspoCART19 cells presented low vector copy numbers and close-to-random vector integration profiles. Moreover, final TranspoCART19 products lacked non-integrated genomic material used for the generation of CAR-T cells and were free from transposase protein.  biodistribution analyses revealed that TranspoCART19 cells were mainly present in hematopoietic organs with no gender bias. Altogether, this study provides a cost-effective, GMP-compliant manufacturing process for the generation of CAR-T cells using non-viral vectors. These results have supported the approval of a clinical trial to evaluate TranspoCART19 cells in patients with relapsed/refractory lymphoma (NCT06378190) that is currently ongoing.

Generation and GMP scale-up of human CAR-T cells using non-viral  transposons for B cell malignances.

Chimeric antigen receptor T cell (CAR-T) therapies have revolutionized cancer immunotherapy. Traditional single-chain variable fragments (ScFvs) used as CAR recognition moieties face challenges such as high tonic signaling, compromised binding epitopes, and suboptimal affinity. Single-domain antibodies (SdAbs) offer an attractive alternative due to their smaller size, stability, and reduced immunogenicity. In this work, we developed an SdAb-CAR-T cell discovery platform integrating generation, characterization, and selection of SdAbs based on various properties. This approach was demonstrated by developing CAR-T cells with SdAbs against CD33, a target for acute myeloid leukemia (AML). We identified diverse SdAbs against CD33, with affinities ranging from 3.9-115 nM, and characterized their binding kinetics and epitope recognition. Using SdAb-based second-generation CARs, we assessed tonic signaling, T cell phenotypes, cytotoxicity and cytokine release , resulting in reduced tonic signaling and increased cytokine production. , SdAb-based CAR-T cells exhibited enhanced efficacy at lower doses, in a xenograft AML mouse model, demonstrating advantages over ScFv-based CD33 CAR-T cells.

Article	Year
Generation of an induced pluripotent stem cell line (CIMAi001-A) from a compound heterozygous Primary Hyperoxaluria Type I (PH1) patient carrying p.G170R and p.R122* mutations in the AGXT ge... Stem cell research\| PubMed ID: 31715429	2019
CRISPR-Cas9 inhibition of hepatic LDH as treatment of primary hyperoxaluria. Molecular therapy. Methods & clinical development\| PubMed ID: 35402636	2022
CAR density influences antitumoral efficacy of BCMA CAR T cells and correlates with clinical outcome. Science advances\| PubMed ID: 36179026	2022
Optimization of universal allogeneic CAR-T cells combining CRISPR and transposon-based technologies for treatment of acute myeloid leukemia. Frontiers in immunology\| PubMed ID: 37795087	2023
Molecular mechanisms promoting long-term cytopenia after BCMA CAR-T therapy in multiple myeloma. Blood advances\| PubMed ID: 39058976	2024
Generation and GMP scale-up of human CAR-T cells using non-viral transposons for B cell malignances. Molecular therapy. Methods & clinical development\| PubMed ID: 40034423	2025
Discovery and preclinical development of a SdAb-based CAR-T technology for targeting CD33 in AML. Molecular therapy. Oncology\| PubMed ID: 40084273	2025

Saray Rodriguez-Diaz

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