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Challenges for adoptive T-cell therapy and proposed gene transfer and genome-editing solutions. Several challenges need to be faced to fully exploit the ability of T lymphocytes to eradicate cancer cells. Several of these challenges can be addressed by gene transfer (gene addition) and genome editing (gene deletion, gene substitution) of  T cells.21 We can redirect the specificity of T cells by inserting, with viral or nonviral vectors, genes encoding for chimeric antigen receptors (CARs) or for T-cell receptors (TCRs) specific for antigens expressed by cancer cells, or we can transfer genes encoding for hybrid receptors, combining pros of CARs and TCRs, such as T-cell receptor fusion constructs (TRuCs)89 or HLA-independent T-cell receptors (HIT receptors),90 synthetic TCRs and antigen receptors (STARs),91 or synthetic chimeric T-cell receptor (ChTCR).92 The specificity of T lymphocytes can be fully redirected through TCR gene editing, which includes a step of deletion of both chain genes (TRAC and TRBC) encoding for the endogenous TCR.10,57,63,65 The safety of T cells and the selective control of adverse events can be improved by the insertion of suicide genes, such as thymidine kinase from herpes simplex virus (HSV-TK)6 or inducible caspase 9 (iCas9),22 CD20, or a truncated form of the epidermal growth factor (EGFR). Recently, more sophisticated synthetic biology approaches, based on logic-gate strategies, Syn/Notch,125 or synthetic intramembrane proteolysis receptors (SNIPRs),126 have been designed to better localize the action of engineered T cells, thus increasing their safety profile.91,125 We can increase the ability of T cells to reach tumor sites by introducing genes encoding for chemokines or chemokine receptors127 or enzymes able to digest and remodel the tumor microenvironment.128 We can make T cells resistant to the immunosuppressive signals present in the tumor microenvironment (TME) by deleting genes encoding for inhibitory receptors,8,58,81-88 or by inserting dominant negative receptors129 or switch receptors,129 able to transform an inhibitory signal into an activating one. We can increase the fitness, function, and persistence of T cells by expressing homeostatic cytokines21 or deleting genes interfering with their persistence and function21 or by adding a chimeric costimulatory receptor (CCR) to engineered cells.
Published: 2025
Figure 1. Challenges for adoptive T-cell therapy and proposed gene transfer and genome-editing solutions. Several challenges need to be faced to fully exploit the ability of T lymphocytes to eradicate cancer cells. Several of these challenges can be addressed by gene transfer (gene addition) and... More about this image found in Challenges for adoptive T-cell therapy and proposed gene transfer and genom...
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Timeline of adoptive T-cell therapy with engineered T cells. This figure represents a timeline of some of the key milestones in adoptive T-cell therapy with engineered cells for cancer treatment. ADA SCID, adenosine deaminase severe combined immunodeficiency; allo-HSCT, allogeneic hematopoietic stem cell transplantation; CAR, chimeric antigen receptors; CMV, cytomegalovirus; DLI, donor lymphocyte infusion; EBV, Epstein-Barr virus; LV, lentiviral vector; mHAg, minor histocompatibility antigens; RV, retroviral vector; TCR, T-cell receptor; TIL, tumor-infiltrating lymphocyte; ZFN, zinc finger nuclease.
Published: 2025
Figure 2. Timeline of adoptive T-cell therapy with engineered T cells. This figure represents a timeline of some of the key milestones in adoptive T-cell therapy with engineered cells for cancer treatment. ADA SCID, adenosine deaminase severe combined immunodeficiency; allo-HSCT, allogeneic hema... More about this image found in Timeline of adoptive T-cell therapy with engineered T cells. This figure r...
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TCRs and CARs. This picture depicts the advantages (green arrow) and disadvantages (red arrow) of chimeric antigen receptors (CARs) and T-cell receptors (TCRs) for adoptive T-cell therapy for cancer. HLA, human leukocyte antigen.
Published: 2025
Figure 3. TCRs and CARs. This picture depicts the advantages (green arrow) and disadvantages (red arrow) of chimeric antigen receptors (CARs) and T-cell receptors (TCRs) for adoptive T-cell therapy for cancer. HLA, human leukocyte antigen. More about this image found in TCRs and CARs. This picture depicts the advantages (green arrow) and disad...
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Genome-editing tools: mode of action. Several genome-editing tools, with different characteristics and modes of action, have been developed and tested for their efficiency in engineering the genome of mammalian cells, including T lymphocytes. While zinc finger nucleases, TALEN, and CRISPR/Cas9 promote gene disruption and/or gene correction or gene substitution through the generation of DNA double-strand break (DSB), base editors and prime editors modify the genome in the absence of DNA DSB. Epigenome editing tools induce transcription inhibition or activation. Depending on the mechanisms of action, the different tools are characterized by a variable risk of genotoxicity (translocation between the different DNA DSB sites, off-target base insertion and/or deletions [INDELs], large deletions, or delayed proliferation through p53 activation).155 Cas9, caspase 9; pegRNA, prime editing guide RNA; sgRNA, single guide RNA; UGI, uracil DNA glycosylase inhibitor.
Published: 2025
Figure 4. Genome-editing tools: mode of action. Several genome-editing tools, with different characteristics and modes of action, have been developed and tested for their efficiency in engineering the genome of mammalian cells, including T lymphocytes. While zinc finger nucleases, TALEN, and CRI... More about this image found in Genome-editing tools: mode of action. Several genome-editing tools, with d...
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Manufacturing disease-specific engineered T-cell–based cellular products. Based on disease diagnosis, antigen repertoire, and knowledge of the characteristics of the tumor microenvironment, different biotechnological reagents can be combined to generate T-cell–based cellular products with the optimal safety and efficacy profile. Different transgenes, encoding for tumor-specific TCRs and/or CARs, cytokines (CKs) or chemokine receptors (CCRs), and/or suicide genes, can be transferred on T cells through different vectors, such as lentiviral (LV), adeno-associated (AAV), and integrase defective lentiviral (IDLV) vectors. Several genes encoding for the endogenous TCR repertoire (TRAC and TRBC) or for inhibitory molecules (IRs) can be genetically disrupted in T cells by genome-editing tools (ie, target-specific guide RNAs used with CRISPR/Cas9, base, or prime editors) to increase the fitness, expansion, and/or survival of T cells. All these reagents can be implemented in manufacturing protocols apt to generate T-cell products with a stem memory/central memory phenotype, starting from autologous or allogeneic T cells. ATMP, advanced therapeutic medicinal product; GMP, good medical practice; KO, knock-out; PBMC, peripheral blood mononuclear cell; QC, quality controls.
Published: 2025
Figure 5. Manufacturing disease-specific engineered T-cell–based cellular products. Based on disease diagnosis, antigen repertoire, and knowledge of the characteristics of the tumor microenvironment, different biotechnological reagents can be combined to generate T-cell–based cellular products w... More about this image found in Manufacturing disease-specific engineered T-cell–based cellular products. ...
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