Journal: Journal for Immunotherapy of Cancer

Article Title: T cell-specific non-viral DNA delivery and in vivo CAR-T generation using targeted lipid nanoparticles

doi: 10.1136/jitc-2025-011759

Figure Lengend Snippet: NCtx-CD19 generates stable and functional CAR-T cells in vitro. ( a ) Schematic representation of NCtx, a targeted LNP platform for T cell engineering. The LNP (1) encapsulates mcDNA encoding a CAR or another GOI flanked by SB100x-compatible ITRs (2), along with SB100x transposase mRNA (3) to mediate genomic integration of the GOI upon transient expression of the transposase mRNA. The vehicle is functionalized with anti-CD7 VHH (4) and anti-CD3 scFv (5) targeting ligands. ( b, c ) CD3/CD28 bead-activated T cells were treated with a dose of 800 ng total nucleic acid using NCtx-CD19, surrogate particles lacking SB100x mRNA transposase (−SB100x) or left untreated (untransfected) for 4 days. Cells were monitored over 20 days to evaluate stable genomic integration. ( b ) Transfection efficiency as the percentage of CD19 CAR+T cells as measured by flow cytometry at various time points post-transfection. n=9 different T cell donors, data represent the mean±SD. ( c ) Representative flow plots at day 14 are shown. ( d ) Antigen-specific cytotoxicity of NCtx-CD19-engineered CAR-T cells, measured via luciferase-based killing assays against CD19+ (Nalm6, Raji) and CD19-negative (K562) target cells. CAR-T cells were collected 13 days post-transfection and co-cultured with target cells at varying effector-to-target (E:T) ratios for 24 hours. Specific lysis was calculated relative to untransfected control cells. n=9 using different T cell donors, data represent the mean±SD. ( e ) Antigen-specific cytokine release by NCtx-CD19-engineered CAR-T cells, measured as the secretion of proinflammatory cytokines IL-2 and IFN-γ. Cytokines were measured after a 24-hour co-culture of effector and target cells at an E:T ratio of 2.5:1, using both CAR+ and untransfected T cells. n=1, measured in technical duplicates. ( f ) Transfection efficiency of resting (− pre-activation) and bead-activated (+ pre-activation) T cells at various time points post-transfection as measured by flow cytometry analysis of CD19-CAR+cells. n=2 T cell donors in two independent experiments, data are presented as mean±SD. P values were calculated using two-way ANOVA, mixed effect model with Tukey correction for multiple comparisons ( b, d, f ) or unpaired t-test ( e ). Significance is plotted with ns for p>0.0322 and *p<0.0332, **p<0.0021, ***p<0.0002 and ****p<0.0001. In ( d ) Nalm6 and Raji (CD19+) are compared with K562 (CD19−). ANOVA, analysis of variance; CAR, chimeric antigen receptor; GOI, gene of interest; IFN-γ, interferon-gamma; IL-2, interleukin 2; ITR, inverted terminal repeat; LNP, lipid nanoparticle; mcDNA, minicircle DNA; mRNA, messenger RNA; ns, not significant; scFv, single-chain variable fragment; SSC, side scatter; VHH, variable domain of a heavy-chain-only antibody.

Article Snippet: CD19 CAR expression in in vitro experiments was detected using CD19 CAR detection reagent (Miltenyi Biotec, REA1297, 1:400) and streptavidin-APC-Cy7 (BD, REA746, 1:200), CD19/CD22 dual CAR expression was detected using CD22 CAR detection reagent (Miltenyi Biotec, 130-126-727, 1:1000) and streptavidin-APC-Cy7 (BD, REA746, 1:200).

Techniques: Functional Assay, In Vitro, Expressing, Transfection, Flow Cytometry, Luciferase, Cell Culture, Lysis, Control, Co-Culture Assay, Activation Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: T cell-specific non-viral DNA delivery and in vivo CAR-T generation using targeted lipid nanoparticles

doi: 10.1136/jitc-2025-011759

Figure Lengend Snippet: NCtx-CD19 generates CAR-T cells in vivo resulting in sustained tumor control across a range of doses in a human PBMC model of leukemia. ( a ) Schematic representation of study timeline: NXG mice were injected intravenously with 5×10 5 luciferase-expressing Nalm6 tumor cells, followed by humanization with 5×10 6 huPBMCs. NCtx-CD19 or a NCtx surrogate encapsulating eGFP mcDNA and SB100x mRNA (vehicle control) were administered intravenously at a total nucleic acid dose of 0.5 mg/kg. ( b ) Transfection efficiency in circulating T cells was measured over time via flow cytometry, represented as CAR+ cells/μL of blood. n=4 (NCtx-CD19) or n=5 (vehicle control), data are mean with individual values plotted. ( c ) CD19 CAR mcDNA expression was assessed by flow cytometry in spleen and bone marrow at the study’s end. Vehicle-treated mice reached HEP and were analyzed between days 24–28 (n=2), while NCtx-CD19-treated mice were all terminated and analyzed on day 31 (n=4). Data are presented as mean±SD. ( d ) Nalm6 tumor burden was monitored by BLI. ( e ) Kaplan-Meier survival analysis. Mouse death in the NCtx-CD19 group on day 24 post-Nalm6 was due to anesthetic overdose, unrelated to health or tumor burden. n=5. ( f ) Schematic representation of the dose de-escalation study timeline: NXG mice were humanized with 5×10 6 huPBMCs 11 days pre-LNP injection, followed by intravenous injection with 5×10 5 luciferase-expressing Nalm6 tumor cells 8 days before treatment. NCtx-CD19 or a NCtx vehicle control encapsulating only SB100x mRNA (vehicle control) were administered intravenously at a dose of total nucleic acid of either 40 or 4 µg/kg. ( g ) Nalm6 tumor burden was monitored via BLI. P values were calculated using two-way ANOVA, mixed effect model ( b ), unpaired t-test ( c ) or log-rank Mantel-Cox test ( e ). Significance is plotted *p<0.0332, **p<0.0021 and ***p<0.0002. ANOVA, analysis of variance; BLI, bioluminescent imaging; CAR, chimeric antigen receptor; HEP, humane endpoint; LNP, lipid nanoparticle; mcDNA, minicircle DNA; mRNA, messenger RNA; PBMC, peripheral blood mononuclear cell; tLNP, targeted lipid nanoparticle.

Article Snippet: CD19 CAR expression in in vitro experiments was detected using CD19 CAR detection reagent (Miltenyi Biotec, REA1297, 1:400) and streptavidin-APC-Cy7 (BD, REA746, 1:200), CD19/CD22 dual CAR expression was detected using CD22 CAR detection reagent (Miltenyi Biotec, 130-126-727, 1:1000) and streptavidin-APC-Cy7 (BD, REA746, 1:200).

Techniques: In Vivo, Control, Injection, Luciferase, Expressing, Transfection, Flow Cytometry, Imaging

Journal: Journal for Immunotherapy of Cancer

Article Title: T cell-specific non-viral DNA delivery and in vivo CAR-T generation using targeted lipid nanoparticles

doi: 10.1136/jitc-2025-011759

Figure Lengend Snippet: NCtx-dual induces robust and durable in vivo CAR-T generation, tumor control and extended survival in CD34+ HSC-engrafted NCG mice. ( a ) Schematic representation of study design: NCG mice engrafted with CD34+ HSC (NCG-His) were injected intravenously with 5×10 5 luciferase-expressing Nalm6 tumor cells, followed by IP injection of 200 ng IL-7. Mice were treated intravenously with NCtx-dual or a NCtx vehicle control encapsulating eGFP mcDNA and SB100x mRNA (vehicle control) at a total nucleic acid dose of 50 µg/kg. ( b ) CD19/CD22 dual CAR mcDNA expression was assessed by flow cytometry in circulating T cells for 40 days post-NCtx administration. n=12, data are presented as mean with individual values. ( c ) Nalm6 tumor burden was monitored by BLI. ( d ) Kaplan-Meier survival analysis. n=6 (vehicle control) or n=12 (NCtx-dual). ( e ) Expression of the exhaustion marker PD-1 in CAR+ and CAR− T cell populations over time in NCtx-dual-treated mice, analyzed by flow cytometry. n=12, data represent mean±individual values. ( f ) T cell phenotype characterization (Tnaive/Tscm, Tcm, Tem, and Teff) based on CD45RA and CD62L expression in CAR+ and CAR− T cells after NCtx-dual administration. n=12, data represent mean±SD. P values were calculated using log-rank Mantel-Cox test ( b ) or two-way ANOVA, mixed effect model ( d, e ). Significance is plotted with ns for p>0.0332 and *p<0.0332. ANOVA, analysis of variance; BLI, bioluminescent imaging; CAR, chimeric antigen receptor; HSC, hematopoietic stem cell; IL-7, interleukin 7; IP, intraperitoneal; mcDNA, minicircle DNA; mRNA, messenger RNA; ns, not significant; PD-1, programmed cell death protein-1; Tcm, central memory T cell; Teff, effector T cell; Tem, effector memory T cell; Tnaive, naïve T cell; Tscm, stem cell memory T cell.

Article Snippet: CD19 CAR expression in in vitro experiments was detected using CD19 CAR detection reagent (Miltenyi Biotec, REA1297, 1:400) and streptavidin-APC-Cy7 (BD, REA746, 1:200), CD19/CD22 dual CAR expression was detected using CD22 CAR detection reagent (Miltenyi Biotec, 130-126-727, 1:1000) and streptavidin-APC-Cy7 (BD, REA746, 1:200).

Techniques: In Vivo, Control, Injection, Luciferase, Expressing, Flow Cytometry, Marker, Imaging

Journal: STAR Protocols

Article Title: Protocol for detecting substrates in living cells by targeted molecular probes through hyperpolarized 129 Xe MRI

doi: 10.1016/j.xpro.2022.101499

Figure Lengend Snippet:

Article Snippet: 10 mm microimaging probe (MICWB40 RES 400 1 H/ 129 Xe 040/010 LLTR) , Bruker , https://bruker.com.

Techniques: Recombinant, Modification, Synthesized, Software

Journal: STAR Protocols

Article Title: Protocol for detecting substrates in living cells by targeted molecular probes through hyperpolarized 129 Xe MRI

doi: 10.1016/j.xpro.2022.101499

Figure Lengend Snippet: Equipment for cell count and NMR experiment

Article Snippet: 10 mm microimaging probe (MICWB40 RES 400 1 H/ 129 Xe 040/010 LLTR) , Bruker , https://bruker.com.

Techniques: Cell Counting

Journal: iScience

Article Title: Metabolic changes of human induced pluripotent stem cell-derived cardiomyocytes and teratomas after transplantation

doi: 10.1016/j.isci.2024.111234

Figure Lengend Snippet:

Article Snippet: Anti-cardiac troponin T (clone: REA 400) , Miltenyi Biotec , Cat#130-129-225; RRID: AB_2922012.

Techniques: Recombinant, Saline, In Vivo, Software