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

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.

Hepatocytes-like cells (HLC) derived from human induced pluripotent stem cells show great promise for cell-based liver therapies and disease modelling. However, their application is currently hindered by the low production yields of existing protocols. We aim to develop a bioprocess able to generate high numbers of HLC. We used stirred-tank bioreactors with a rational control of dissolved oxygen concentration (DO) for the optimization of HLC production as 3D aggregates. We evaluated the impact of controlling DO at physiological levels (4%O) during hepatic progenitors' stage on cell proliferation and differentiation efficiency. Whole transcriptome analysis and biochemical assays were performed to provide a detailed characterization of HLC quality attributes. When DO was controlled at 4%O during the hepatic progenitors' stage, cells presented an upregulation of genes associated with hypoxia-inducible factor pathway and a downregulation of oxidative stress genes. This condition promoted higher HLC production (maximum cell concentration: 2 × 10 cell/mL) and improved differentiation efficiencies (80% Albumin-positive cells) when compared to the bioreactor operated under atmospheric oxygen levels (21%O, 0.6 × 10 cell/mL, 43% Albumin positive cells). These HLC exhibited functional characteristics of hepatocytes: capacity to metabolize drugs, ability to synthesize hepatic metabolites, and inducible cytochrome P450 activity. Bioprocess robustness was confirmed with HLC derived from different donors, including a primary hyperoxaluria type 1 (PH1) patient. The generated PH1.HLC showed metabolic features of PH1 disease with higher secretion of oxalate compared with HLC generated from healthy individuals. This work reports a reproducible bioprocess, that shows the importance of controlling DO at physiological levels to increase HLC production, and the HLC capability to display PH1 disease features.

Oxygen control in bioreactor drives high yield production of functional hiPSC-like hepatocytes for advanced liver disease modelling.

To explore the role of bone marrow adipocytes (BMAds) in osteoarthritis (OA).

Article	Year
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
Oxygen control in bioreactor drives high yield production of functional hiPSC-like hepatocytes for advanced liver disease modelling. Scientific reports\| PubMed ID: 39427033	2024
Implication of bone marrow adipose tissue in bone homeostasis during osteoarthritis. Osteoarthritis and cartilage\| PubMed ID: 40154729	2025

Maria E. Calleja-Cervantes

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