The piggyBac transposon system provides a non-viral alternative for cost-efficient and simple chimeric antigen receptor (CAR) T cell production. The generation of clinical-grade CAR T cells requires strict adherence to current good manufacturing practice (cGMP) standards. Unfortunately, the high costs of commonly used lentiviral or retroviral vectors limit the manufacturing of clinical-grade CAR T cells in many non-commercial academic institutions. Here, we present a manufacturing platform for highly efficient generation of CD19-specific CAR T cells (CAR19 T cells) based on co-electroporation of linear DNA transposon and mRNA encoding the piggyBac transposase. The transposon is prepared enzymatically in vitro by PCR and contains the CAR transgene flanked by piggyBac 3' and 5' arms. The mRNA is similarly prepared via in vitro transcription. CAR19 T cells are expanded in the combination of cytokines interleukin (IL)-4, IL-7, and IL-21 to prevent terminal differentiation of CAR T cells. The accurate control of vector copy number (VCN) is achieved by decreasing the concentration of the transposon DNA, and the procedure yields up to 1 × 108 CAR19 T cells per one electroporation of 1 × 107 peripheral blood mononuclear cells (PBMCs) after 21 days of in vitro culture. Produced cells contain >60% CAR+ cells with VCN < 3. In summary, the described manufacturing platform enables a straightforward cGMP certification, since the transposon and transposase are produced abiotically in vitro via enzymatic synthesis. It is suitable for the cost-effective production of highly experimental, early-phase CAR T cell products.

• Klíčová slova
• CD19, T cells, chimeric antigenic receptor, electroporation, piggyBac transposon,
• Publikační typ
• časopisecké články MeSH

BACKGROUND AIMS: Clinical-grade chimeric antigenic receptor (CAR)19 T cells are routinely manufactured by lentiviral/retroviral (LV/RV) transduction of an anti-CD3/CD28 activated T cells, which are then propagated in a culture medium supplemented with interleukin (IL)-2. The use of LV/RVs for T-cell modification represents a manufacturing challenge due to the complexity of the transduction approach and the necessity of thorough quality control. METHODS: We present here a significantly improved protocol for CAR19 T-cell manufacture that is based on the electroporation of peripheral blood mononuclear cells with plasmid DNA encoding the piggyBac transposon/transposase vectors and their cultivation in the presence of cytokines IL-4, IL-7 and IL-21. RESULTS: We found that activation of the CAR receptor by either its cognate ligand (i.e., CD19 expressed on the surface of B cells) or anti-CAR antibody, followed by cultivation in the presence of cytokines IL-4 and IL-7, enables strong and highly selective expansion of functional CAR19 T cells, resulting in >90% CAR+ T cells. Addition of cytokine IL-21 to the mixture of IL-4 and IL-7 supported development of immature CAR19 T cells with central memory and stem cell memory phenotypes and expressing very low amounts of inhibitory receptors PD-1, LAG-3 and TIM-3. CONCLUSIONS: Our protocol provides a simple and cost-effective method for engineering high-quality T cells for adoptive therapies.

• Klíčová slova
• CAR T cells, IL-21, IL-4, IL-7, cancer immunotherapy, piggyBac transposon,
• MeSH
• aktivace lymfocytů účinky léků   genetika   MeSH
• buněčné kultury metody   MeSH
• buňky PC-3 MeSH
• chimerické antigenní receptory genetika   metabolismus   MeSH
• elektroporace MeSH
• genetické vektory MeSH
• HEK293 buňky MeSH
• imunoterapie adoptivní metody   MeSH
• interleukin-4 farmakologie   MeSH
• interleukin-7 farmakologie   MeSH
• interleukiny farmakologie   MeSH
• kultivované buňky MeSH
• Lentivirus genetika   MeSH
• lidé MeSH
• proteinové inženýrství metody   MeSH
• protinádorové vakcíny genetika   imunologie   MeSH
• receptory antigenů T-buněk genetika   metabolismus   MeSH
• T-lymfocyty * cytologie   účinky léků   imunologie   metabolismus   MeSH
• transdukce genetická metody   MeSH
• transpozibilní elementy DNA genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chimerické antigenní receptory MeSH
• Interleukin-21 MeSH
• interleukin-4 MeSH
• interleukin-7 MeSH
• interleukiny MeSH
• protinádorové vakcíny MeSH
• receptory antigenů T-buněk MeSH
• transpozibilní elementy DNA MeSH

B-cell lymphoma (BCL) is the most common hematologic malignancy. While sequencing studies gave insights into BCL genetics, identification of non-mutated cancer genes remains challenging. Here, we describe PiggyBac transposon tools and mouse models for recessive screening and show their application to study clonal B-cell lymphomagenesis. In a genome-wide screen, we discover BCL genes related to diverse molecular processes, including signaling, transcriptional regulation, chromatin regulation, or RNA metabolism. Cross-species analyses show the efficiency of the screen to pinpoint human cancer drivers altered by non-genetic mechanisms, including clinically relevant genes dysregulated epigenetically, transcriptionally, or post-transcriptionally in human BCL. We also describe a CRISPR/Cas9-based in vivo platform for BCL functional genomics, and validate discovered genes, such as Rfx7, a transcription factor, and Phip, a chromatin regulator, which suppress lymphomagenesis in mice. Our study gives comprehensive insights into the molecular landscapes of BCL and underlines the power of genome-scale screening to inform biology.

• MeSH
• B-buněčný lymfom genetika   patologie   MeSH
• buněčné klony MeSH
• CRISPR-Cas systémy genetika   MeSH
• genetické asociační studie MeSH
• genetické testování metody   MeSH
• genová dávka MeSH
• geny nádorové MeSH
• lidé MeSH
• myši inbrední C57BL MeSH
• myši transgenní MeSH
• receptory antigenů B-buněk metabolismus   MeSH
• regulace genové exprese u nádorů MeSH
• reprodukovatelnost výsledků MeSH
• transpozibilní elementy DNA genetika   MeSH
• tumor supresorové geny MeSH
• ztráta heterozygozity MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• receptory antigenů B-buněk MeSH
• transpozibilní elementy DNA MeSH

BACKGROUND: The non-viral production of CAR-T cells through electroporation of transposon DNA plasmids is an alternative approach to lentiviral/retroviral methods. This method is particularly suitable for early-phase clinical trials involving novel types of CAR-T cells. The primary disadvantage of non-viral methods is the lower production efficiency compared to viral-based methods, which becomes a limiting factor for CAR-T production, especially in chemotherapy-pretreated lymphopenic patients. METHODS: We describe a good manufacturing practice (GMP)-compliant protocol for producing CD19 and CD123-specific CAR-T cells based on the electroporation of transposon vectors. The lymphocytes were purified from the blood of patients undergoing chemotherapy for B-NHL or AML and were electroporated with piggyBac transposon encoding CAR19 or CAR123, respectively. Electroporated cells were then polyclonally activated by anti-CD3/CD28 antibodies and a combination of cytokines (IL-4, IL-7, IL-21). The expansion was carried out in the presence of irradiated allogeneic blood-derived mononuclear cells (i.e., the feeder) for up to 21 days. RESULTS: Expansion in the presence of the feeder enhanced CAR-T production yield (4.5-fold in CAR19 and 9.3-fold in CAR123). Detailed flow-cytometric analysis revealed the persistence of early-memory CAR-T cells and a low vector-copy number after production in the presence of the feeder, with no negative impact on the cytotoxicity of feeder-produced CAR19 and CAR123 T cells. Furthermore, large-scale manufacturing of CAR19 carried out under GMP conditions using PBMCs obtained from B-NHL patients (starting number=200x10e6 cells) enabled the production of >50x10e6 CAR19 in 7 out of 8 cases in the presence of the feeder while only in 2 out of 8 cases without the feeder. CONCLUSIONS: The described approach enables GMP-compatible production of sufficient numbers of CAR19 and CAR123 T cells for clinical application and provides the basis for non-viral manufacturing of novel experimental CAR-T cells that can be tested in early-phase clinical trials. This manufacturing approach can complement and advance novel experimental immunotherapeutic strategies against human hematologic malignancies.

• Klíčová slova
• CAR-T cells, PiggyBac PB transposon, electroporation, leukemia, lymphoma,
• MeSH
• akutní myeloidní leukemie * terapie   imunologie   genetika   MeSH
• allogeneické buňky imunologie   MeSH
• antigeny CD19 * imunologie   genetika   MeSH
• B-buněčný lymfom terapie   imunologie   genetika   MeSH
• chimerické antigenní receptory * genetika   imunologie   MeSH
• elektroporace MeSH
• imunoterapie adoptivní * metody   MeSH
• lidé MeSH
• podkladové buňky MeSH
• T-lymfocyty imunologie   metabolismus   MeSH
• transpozibilní elementy DNA * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigeny CD19 * MeSH
• chimerické antigenní receptory * MeSH
• transpozibilní elementy DNA * MeSH

[This corrects the article DOI: 10.3389/fimmu.2024.1415328.].

• Klíčová slova
• CAR-T cells, PiggyBac PB transposon, electroporation, leukemia, lymphoma,
• Publikační typ
• tisková chyba MeSH

BACKGROUND: The efficiency of chimeric antigen receptor (CAR) T-cell-based therapies depends on a sufficient expansion of CAR T cells in vivo and can be weakened by intra-tumoral suppression of CAR T cell functions, leading to a failure of therapy. For example, certain B-cell malignancies such as chronic lymphocytic leukemia are weakly sensitive to treatment with CAR T cells. Co-expression of proinflamatory cytokines such as IL-12 and IL-18 by CAR T cells have been shown to enhance their antitumor function. We similarly engineered CAR T cell to co-express IL-21 and studied the effects of IL-21 on CAR T cells specific to CD19 and prostate-specific membrane antigens using an in vitro co-culture model and NSG mice transplanted with B-cell tumors. RESULTS: IL-21 enhanced the expansion of CAR T cells after antigenic stimulation, reduced the level of apoptosis of CAR T cells during co-culture with tumor cells and prevented differentiation of CAR T cells toward late memory phenotypes. In addition, induced secretion of IL-21 by CAR T cells promoted tumor infiltration by CD19-specific CAR (CAR19) T cells in NSG mice, resulting in reduced tumor growth. By co-culturing CAR19 T cells with bone-marrow fragments infiltrated with CLL cells we demonstrate that IL-21 reduces the immunosupressive activity of CLL cells against CAR19 T cells. CONCLUSIONS: CAR19 T cells armed with IL-21 exhibited enhanced antitumor functions. IL-21 promoted their proliferation and cytotoxicity against chronic lymphocytic leukemia (CLL). The results suggest that arming CAR T cells with IL-21 could boost the effectiveness of CAR T-mediated therapies.

• Klíčová slova
• CAR T cells, chronic lymphocytic leukemia, interleukin 21, piggyBac transposon,
• MeSH
• fenotyp MeSH
• imunosupresivní léčba MeSH
• interleukiny metabolismus   MeSH
• lidé MeSH
• myši MeSH
• nádory imunologie   terapie   MeSH
• proliferace buněk MeSH
• receptory antigenů T-buněk metabolismus   MeSH
• T-lymfocyty imunologie   MeSH
• transpozibilní elementy DNA genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CD19-specific chimeric antigen receptor MeSH Prohlížeč
• Interleukin-21 MeSH
• interleukiny MeSH
• receptory antigenů T-buněk MeSH
• transpozibilní elementy DNA MeSH

Germline transformation with new transposon vectors now enables causal tests of gene function via ectopic protein expression or RNA interference in non-drosophilid insects. The problem remains of how to drive the transgene expression in vivo. We employed germline transformation using the piggyBac 3xP3-EGFP vector to test whether the Drosophila heat shock hsp70 promoter will be active in the live silkworm. We modified the original vector by cloning the coding sequence for Bombyx nuclear receptor Ftz-F1 between the hsp70 promoter and the terminator. Three independent transgenic lines expressing the Pax-6-driven EGFP marker in larval and adult photoreceptors were obtained with efficiencies of up to 1.7% of fertile G0 adults that gave GFP-positive progeny. Chromosomal integration of the transposon was confirmed with inverse PCR. Heat induction of the transgenic BmFtz-F1 was proven at both the mRNA and protein levels. RT-PCR data showed that the Drosophila heat shock promoter was functional in all three transgenic lines. Although basal activity was apparent at 25 degrees C, 1 h at 42 degrees C induced BmFtz-F1 mRNA at different stages of development and in diverse tissues. The relative levels of induction differed among the transgenic lines. Northern blot hybridization detected transgenic BmFtz-F1 only after heat shock and low levels of the mRNA were still present 6 h after the heat treatment. Immunostaining of epidermis using anti-BmFtz-F1 antibody showed a clear increase of nuclear signal 90 min after a heat shock.

• MeSH
• aktivace transkripce * MeSH
• bourec genetika   MeSH
• DNA vazebné proteiny biosyntéza   chemie   genetika   MeSH
• hmyzí proteiny MeSH
• homeodoménové proteiny MeSH
• klonování DNA MeSH
• messenger RNA genetika   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• proteiny tepelného šoku HSP70 genetika   MeSH
• receptory cytoplazmatické a nukleární MeSH
• sekvence aminokyselin MeSH
• steroidogenní faktor 1 MeSH
• transformace genetická MeSH
• transgeny genetika   MeSH
• transkripční faktory fushi tarazu MeSH
• transkripční faktory biosyntéza   chemie   genetika   MeSH
• vysoká teplota * MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH
• Názvy látek
• DNA vazebné proteiny MeSH
• hmyzí proteiny MeSH
• homeodoménové proteiny MeSH
• messenger RNA MeSH
• nuclear hormone receptor FTZ-F1, Bombyx mori MeSH Prohlížeč
• proteiny tepelného šoku HSP70 MeSH
• receptory cytoplazmatické a nukleární MeSH
• steroidogenní faktor 1 MeSH
• transkripční faktory fushi tarazu MeSH
• transkripční faktory MeSH

Multiple myeloma (MM) has witnessed improved patient outcomes through advancements in therapeutic approaches. Notably, allogeneic stem cell transplantation, proteasome inhibitors, immunomodulatory drugs, and monoclonal antibodies have contributed to enhanced quality of life. Recently, a promising avenue has emerged with chimeric antigen receptor (CAR) T cells targeting B-cell maturation antigen (BCMA), expressed widely on MM cells. To mitigate risks associated with allogenic T cells, we investigated the potential of BCMA CAR expression in natural killer cells (NKs), known for potent cytotoxicity and minimal side effects. Using the NK-92 cell line, we co-expressed BCMA CAR and soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) employing the piggyBac transposon system. Engineered NK cells (CAR-NK-92-TRAIL) demonstrated robust cytotoxicity against a panel of MM cell lines and primary patient samples, outperforming unmodified NK-92 cells with a mean difference in viability of 45.1% (±26.1%, depending on the target cell line). Combination therapy was explored with the proteasome inhibitor bortezomib (BZ) and γ-secretase inhibitors (GSIs), leading to a significant synergistic effect in combination with CAR-NK-92-TRAIL cells. This synergy was evident in cytotoxicity assays where a notable decrease in MM cell viability was observed in combinatorial therapy compared to single treatment. In summary, our study demonstrates the therapeutic potential of the CAR-NK-92-TRAIL cells for the treatment of MM. The synergistic impact of combining these engineered NK cells with BZ and GSI supports further development of allogeneic CAR-based products for effective MM therapy.

• Klíčová slova
• allogenic, cancer, chimeric antigen receptor, immunotherapy, multiple myeloma, natural killer,
• MeSH
• bortezomib farmakologie   terapeutické užití   MeSH
• chimerické antigenní receptory * metabolismus   MeSH
• imunoterapie adoptivní MeSH
• kvalita života MeSH
• lidé MeSH
• maturační antigen B-buněk metabolismus   MeSH
• mnohočetný myelom * patologie   terapie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bortezomib MeSH
• chimerické antigenní receptory * MeSH
• maturační antigen B-buněk MeSH