Application of Transposon Systems in The Transgenesis of Bovine Somatic And Germ Cells

Background: Several DNA transposons, PiggyBac (PB), Sleeping beauty (SB) and Tol2 have been applied as effective means for transgenesis in many species. Cattle are not typical experimental animals, and relatively little verification has been studied in this species. Thus, the goal of this study was the applicability of three transposon systems in somatic and embryo cells in cattle, while also determining which of the three systems is appropriate for each type of cell. To conduct the experiment, green fluorescent protein (GFP)-expressing transposon systems were used for electroporation and microinjection in the somatic cells and embryo stage, respectively. After transfection, GFP-positive cells or blastocysts were observed through a fluorescent microscope and transfection efficiency was calculated by FACS.Results: In the bovine somatic cells experiment, the PB (63.97 ± 11.56) showed higher efficiency as compared to the other two systems (SB: 50.74 ± 13.02 and Tol2: 16.55 ± 5.96). Unlike the results of the somatic cells, Tol2 (75.00%) and SB (70.00%) in the embryo were more efficient as compared to PB (42.86 %).Conclusions: These results demonstrate that all three transposon systems can be used in bovine somatic cells and embryos as a gene engineering experimental method and which type of transposon system is appropriate to apply depending on the cell type.

Caperucita y la abuela-lobo: Los cuentos de hadas queer de Alejandra Pizarnik

Near the end of her life, the Argentine poet Alejandra Pizarnik (1936-1972) wrote a series of short prose works that explicitly rewrite fairy tales or take up their atmosphere and some of their elements. Such is the case of “Violario” (1971), which rewrites Little Red Riding Hood, with an emphasis on the link between violence and sexuality, and “A tiempo y no” (1968) which highlights the most sinister elements of Snow White. This last story, along with Sleeping Beauty, The Little Mermaid and Hansel and Gretel are also rewritten in prose pieces, poems and in her Diaries. This article analyzes how these rewritings take place—starting with the convergences and divergences with the hypotexts—to illuminate the anomalous and queer image of childhood created by Pizarnik. --- La poeta argentina Alejandra Pizarnik (1936-1972) hacia el final de su vida escribe una serie de prosas breves que recuperan explícitamente cuentos de hadas, o retoman su atmósfera y algunos de sus elementos. Tal es el caso de “Violario” (1971), que reescribe Caperucita roja, con un marcado énfasis en el cruce entre violencia y sexualidad, y “A tiempo y no” (1968) que retoma y subraya los elementos más siniestros de Blancanieves. Este último cuento, junto con La bella durmiente, La sirenita y Hansel y Gretel son recuperados también en otras prosas, poemas y en los Diarios. Indagar el modo en que tienen lugar estas reescrituras, a partir de las convergencias y divergencias con sus hipotextos, iluminará zonas de la infancia anómala y queer que Pizarnik configura.

Sleeping Beauty: Anesthesia May Promote Relapse in Dogs With Diffuse Large B-Cell Lymphoma in Complete Remission After Chemo-Immunotherapy

Surgery-induced stress and anesthesia-related immunosuppression are believed to play a critical role in human oncology patients. Studies have hypothesized that anesthesia influences patients' outcome, promoting tumor recurrence and metastasis. Aim of the study was to investigate whether anesthesia promoted relapse in dogs with diffuse large B-cell lymphoma (DLBCL). Medical records were searched for dogs with DLBCL, that were in complete remission (CR) after the same chemo-immunotherapy protocol. Dogs receiving anesthesia were included if the procedure was performed while in CR. Time to relapse (TTR) was obtained via Kaplan–Meier method. Association between anesthesia and relapse was assessed using a nested case-control design and estimated using conditional logistic regression. Sixty-one dogs with DLBCL were included. Overall median TTR was 329 days (95% CI, 281–377). Forty-eight (79%) dogs relapsed during the study period, while 13 (21%) were still in CR at data analysis closure. Eighteen (30%) dogs received anesthesia with opioids, propofol, and isoflurane or sevoflurane. The relative risk of lymphoma relapse for dogs undergoing anesthesia was significantly higher compared with dogs not undergoing anesthesia, with an odds ratio of 3.09 (P = 0.019) on multivariable analysis. Anesthesia may promote relapse in dogs with DLBCL treated with chemo-immunotherapy, although a role of perioperative stress cannot be ultimately excluded. Considering the high frequency of anesthetic procedures required for diagnostic and therapeutic protocols among oncology patients, it is of utmost interest to characterize the effects of single anesthetic agents on the immune system. Further prospective studies are needed to better define the impact of anesthesia on patients' outcome.

Music as Formal and Signifying Feature in García Márquez’s Mature Fiction

Music has played a varying role in García Márquez’s work since the passing reference to traveling troubadour “Francisco el Hombre” in One Hundred Years of Solitude and the novel’s concealed presence of Colombian vallenato song. In The Autumn of the Patriarch music becomes more prominent, with such musical traits as romantic bolero formulas, quotations from folk tunes, children’s jingles, and allusions to Caribbean pop rhythms. These musical insertions help provide markers to the relentless verbal flow of the work. In addition, the larger form of the novel is, by admission of the author, modeled after the string quartets of Bela Bartok. Classical music, moreover, contributes some black humor, as in the refined, cultured thug José Ignacio Sáenz de la Barra’s attachment to Mozart and Bruckner. A vallenato serves as the epigraph to Love in the Time of Cholera and also foreshadows crucial events. Musical references, moreover, furnish chronological and character markers in that novel, with Florentino and Juvenal employing music to captivate Fermina, the first communicating directly with his self-trained violin playing and the other hiring a professional to perform on a grand piano under her balcony. During their one encounter, the two men turn from the economic issue at hand to a discussion of music. García Márquez’s last novel fuses Florentino and Juvenal into a modernist musical voice in his narration of Memories of My Melancholy Whores. Punctuating his recollections with “high art” musical allusions, he also communicates directly by singing a medieval Spanish ballad to his sleeping beauty.

Implication of ICOSLG on Relapse in Infant T(4;11) Acute Lymphoblastic Leukemia

Infant t(4;11) acute lymphoblastic leukemia (ALL) is associated with a high relapse rate with 4-year event-free survival (EFS) of only 36%. Relapse has been shown to be the major cause of death as 83% of relapsed infant t(4;11) ALL patients die within three years of diagnosis. Therefore, it is of utmost therapeutic interest to elucidate molecular mechanisms of relapse. Here we show that t(4;11) ALL cells upregulate the inducible T-cell costimulator ligand (ICOSLG) in an early growth response 3 (EGR3) dependent manner thereby promoting the development of regulatory T-cells (T regs). We propose that short EFS and high ICOSLG expression are causally linked, in that ICOSLG-mediated induction of T regs interferes with immune recognition of leukemia cells. According to that hypothesis, ICOSLG would not only be a novel and independent prognostic biomarker but a potential therapeutic target. We investigated the function of EGR3 in infant ALL since EGR3 has been described as an indirect target of the iroquois homeobox 1 (IRX1) transcription factor. For that purpose we created a stable sleeping beauty transposon-based SEM cell line expressing EGR3 in a Doxycycline-inducible manner. Gene expression and western blot analysis revealed strong upregulation of ICOSLG 48h after Doxycycline induction when compared to an empty vector control. Additionally, chromatin immunoprecipitation (ChIP) experiments depicted that EGR3 directly binds to the promoter of the ICOSLG gene. The expression of ICOSLG in mesenchymal stem cells has been shown to foster the induction of T regs and ICOSLG-mediated T reg expansion has been identified as a driver of acute myeloid leukemia and glioblastoma. In the bone marrow (BM) hematopoietic stem cells (HSC) colocalize with T regs which provide an immune privilege to the stem cell niche. Based on these observations, we hypothesize that ICOSLG expressing ALL cells could create an immune privileged niche appearing to be necessary for HSC maintenance and sanctuary from immune attack. This could create independence from the BM immune privilege enabling migration of the ALL cells. If so, this could contribute to minimal residual disease (MRD) formation after induction therapy and subsequently to higher probability of relapse. To evaluate our hypothesis, we cocultured the EGR3-SEM and control cells with primary human CD4 + and CD8 + T-cells. The T-lymphocytes were isolated from the peripheral blood mononuclear cells (PBMC) of healthy donors and stimulated with coated α-CD2, -CD3 and -CD28 beads. We observed 16% more CD4 +CD25 ++FOXP3 + T regs after 48h of coculture with EGR3-SEM cells compared to the control. The addition of a neutralizing monoclonal α-ICOSLG antibody to the coculture led to a reduction of the T reg frequency in EGR3-SEM coculture. Taken together, these results strongly suggest that ALL cells expressing EGR3 induce the formation of T regs via ICOSLG expression. To confirm our results with patient-derived material, we investigated the gene expression of 50 infant t(4;11) pro-B phenotypic ALL patients. Pearson correlation testing confirmed that ICOSLG expression strongly correlates with EGR3 and IRX1 expression. Furthermore, we were able to classify the patients considering their ICOSLG expression level into an ICOSLG-high (ICOSLG-hi) and an ICOSLG-low (ICOSLG-lo) group. Outcome data for a 5-year follow-up were available for 35 of 50 patients, n=5 ICOSLG-hi and n=30 ICOSLG-lo. 100% (5/5) of the ICOSLG-hi patients failed within 17 months of diagnosis whereas 53% (16/30) of the ICOSLG-lo patients failed within 56 months of diagnosis. The remaining 14 ICOSLG-lo patients were alive after 60 months. These data underscore the role of overexpressed ICOSLG in relapse development. However, a verification of these findings in a larger cohort is needed. In conclusion, our study identifies ICOSLG as a promising prognostic marker and novel therapeutic target in infant t(4;11) ALL. Furthermore, our findings implicate the interaction between T-cells and leukemia stem cells as contributory to disease progression. Disclosures Cario: Novartis: Other: Lecture Fee.

Donor-Derived CAR T Cells Engineered with Sleeping Beauty in Pediatric and Adult Patients with Acute Lymphoblastic Leukemia Relapsed Post-HSCT

Introduction Allogeneic Chimeric Antigen Receptor (CAR) T cells engineered with non-viral methods offer a modality to reduce costs and logistical complexity of the viral process and allow lymphodepleted patients to access CAR T cell treatment. We recently proposed the use of Sleeping Beauty (SB) transposon to engineer donor-derived T cells differentiated according to the cytokine-induced killer (CIK) cell protocol (Magnani CF et al. J Clin Invest. 2021). We report here outcomes on B-cell acute lymphoblastic leukemia (B-ALL) patients, relapsing after transplantation, treated with donor-derived anti-CD19 CAR T cells (CARCIK-CD19). Methods We conducted an academic, multi-center, phase I/II dose-escalation trial in patients relapsed after allogeneic hematopoietic stem cell transplantation (HSCT). The infusion product was manufactured in-house starting from 50 mL of peripheral blood from the HSCT donor by electroporation with GMP-grade plasmids. All patients underwent lymphodepletion with Fludarabine (30 mg/m 2/day x 4 days) and Cyclophosphamide (500 mg/m 2/day x 2 days), before proceeding to CARCIK-CD19 infusion. We used the Bayesian Optimal Interval (BOIN) design to define a four-dose escalation scheme. Primary objectives were to define the Maximum Tolerated Dose (MTD), safety, and feasibility. Secondary objectives included the assessment of complete hematologic response (CR), duration of response (DOR), progression-free (PFS), event-free (EFS), and overall survival (OS). This study was registered at ClinicalTrials.gov, NCT03389035. Results From January 2018 to June 2021, a total of 32 patients were screened, 26 enrolled (6 children and 20 adults) and 21 infused (4 children and 17 adults). Reasons for not receiving infusion included consent withdrawal (N=1), disease progression not controlled by bridging therapy (N=3), acquisition of myeloid phenotype (N=1). The median number of prior therapies was 4 (range, 1-7) with a median time interval from HSCT to relapse of 9 months. The median BM blasts was 60% (range, 5-100%) at enrollment and 7% (range, 0-96%) post lymphodepletion. Of the 21 patients infused, CARCIK-CD19 were obtained by HLA-identical sibling (n=6, 29%), matched unrelated (n= 7, 33%), and haploidentical donors (n=8, 38%). Three patients (14%) received the first dose level of 1x10 6 CARCIK-CD19 cells/Kg, three (14%) the second of 3x10 6, and three (14%) the third of 7.5x10 6 whereas 12 patients (57%) received the fourth and last planned dose level of 15x10 6 cells/Kg, as no dose limiting toxicity (DLT) was observed. CRS was observed in six patients (three grade I and three grade II) and immune effector cell-associated neurotoxicity in two patients at the highest dose. Although 9 out of 21 had experienced acute or chronic graft-versus-host disease (GvHD) after the previous HSCT, secondary GvHD was never induced by CARCIK-CD19. Complete response was achieved by 13 out of 21 patients (61.9%, 95%CI=38-82%) and by 11 out of 15 patients treated with the 2 highest doses (73.3%, 95%CI=45-92%). Eleven of these responders were MRD-negative. Notably, the type of donor did not influence the achievement of CR 28 days post-infusion. At a median follow up of 21.6 months (range, 1.0-38.4 months), 10 patients (47.6%) are alive in CR (9 in the 2 highest dose levels). Overall, the median OS and EFS were 9.7 and 3.2 months, respectively, with a median DOR of 4.0 months (range, 1.0-23.5 months). Patients in CR at 28-days had a 6-months relapse-free survival of 48.4% (SE=14.9). EFS at 6 months was 26.5% (SE=9.9) and OS was 67.6% (SE=11.1). Among the 13 patients who achieved CR, two children underwent consolidation with a second allo-HSCT in complete remission. Adult patients did not receive any additional anti-leukemic therapies unless a relapse occurred, and four of them remained in remission and alive (+24, +9, +6, and +4 months). Robust CARCIK-CD19 cell expansion was achieved in most patients and CARCIK-CD19 cells were measurable for up to 22 months. Conclusions SB-engineered CAR T cells induce sustained responses in B-ALL patients relapsed after HSCT irrespective of the donor type and without severe toxicities. Disclosures Lussana: Incyte: Honoraria; Pfizer: Honoraria; Astellas Pharma: Honoraria; Amgen: Honoraria. Gritti: Takeda: Consultancy; Roche: Consultancy; Kite Gilead: Consultancy; IQvia: Consultancy; Italfarmaco: Consultancy; Clinigen: Consultancy. Biondi: Incyte: Consultancy, Other: Advisory Board; Bluebird: Other: Advisory Board; Novartis: Honoraria; Amgen: Honoraria; Colmmune: Honoraria.

Persistent Control of SIV Infection in Rhesus Macaques By Expressing a Highly Potent SIV Decoy Receptor after In Vivo HSC Transduction

Despite the success achieved by antiretroviral HIV-1 therapies, long-term and repeated drug administration is associated with toxicity, virus evasion and high cost. We aim to develop a gene therapy approach for persistent control against HIV-1 infection by delivering the transgene expressing a decoy receptor (eCD4-Ig) to hematopoietic stem cells (HSCs) by in vivo transduction. In this approach, HSCs are mobilized from the bone marrow into the peripheral blood stream and transduced with intravenously injected virus vectors. We use an integrating, helper-dependent adenovirus (HDAd5/35++) vector system that targets human CD46, a receptor that is abundantly expressed on primitive HSCs. Transgene integration is achieved by a hyperactive Sleeping Beauty transposase and transgene marking in peripheral blood cells can be increased by in vivo selection. The efficacy and safety of our in vivo HSC transduction/selection strategy has been previously demonstrated for the treatment of b-hemoglobinopathies, hemophilia A, and cancer in murine disease models. In non-human primates, we showed efficient transgene (g-globin) expression in peripheral red blood cells using this strategy. eCD4-Ig functions like a neutralizing antibody and shows broad neutralization spectrum against HIV-1, HIV-2 and SIV isolates. We have designed and produced an HDAd5/35++ vector expressing rhesus eCD4-Ig (rh-eCD4-Ig) from a constitutive and highly active EF1a promoter. In CD46-transgenic mice, over 50µg/mL eCD4-Ig in serum was measured after in vivo transduction and selection, with no obvious adverse events observed. Neutralization assay with serum samples showed that the produced eCD4-Ig effectively inhibited HIV-1 and SIV infection. We then performed studies in a rhesus macaque. After in vivo HSC transduction/selection of a rhesus macaque, eCD4-Ig serum levels were stable at 20-30 mg/ml. In vitro SIVmac239 neutralization assays using week 13 serum and recombinant eCD4-Ig protein determined that the IC50 of eCD4-Ig in rhesus serum is 1.0 mg/ml. This implies that the serum eCD4-Ig concentration at the time of SIVmac239 challenge was ~25-fold higher than the IC50. High-level eCD4-Ig expression had no clinical or hematological side effects. The first SIVmac239 challenge (20pg) was given on June 22 nd. Increasing rechallenge doses are currently injected monthly. So far, the viral load measured by quantitative RT-PCR is below detection limit. The animal is without symptoms and has normal lymphocyte/subset counts. Our study demonstrates an in vivo HSC transduction approach for potential long-term control of HIV-1 infection. Disclosures Kiem: VOR Biopharma: Consultancy; Ensoma Inc.: Consultancy, Current holder of individual stocks in a privately-held company; Homology Medicines: Consultancy. Lieber: Ensoma: Research Funding.

Combining the Expression of CD33.CAR and CXCR4 to Increase CAR-CIK Cell Homing to Bone Marrow Niche and Leukemic Stem Cell Eradication in Acute Myeloid Leukemia

Chimeric Antigen Receptor (CAR) T cell therapy is a promising treatment for acute myeloid leukemia (AML), but a limited efficacy was reported from ongoing clinical trials. The capacity of engineered T cells to infiltrate into the bone marrow (BM) niche, where leukemic stem cells (LSC) reside, strongly impacts the success of the treatment. Ex vivo manipulation of CAR T cells affects the expression of several chemokine receptors and may alter the capacity of infused cells to migrate to BM. The chemokine ligand 12 (CXCL12), released by mesenchymal stromal cells (MSCs) within the medullary niche, and its chemokine receptor 4 (CXCR4) regulate leukocytes trafficking to the BM. In AML, CXCL12 binds CXCR4 over-expressed on blasts, promoting their homing in the niche. CXCR4 expression is drastically downregulated during the culture of cytokine induced killer (CIK) cells, an interesting effector T cell population with acquired NK-like cytotoxicity along with minimal alloreactivity. Therefore, combining the expression of CD33.CAR with the over-expression of CXCR4 might facilitate CAR-CIKs homing to the BM and subsequent leukemia eradication. We designed two bicistronic Sleeping Beauty transposon vectors: CXCR4(IRES)CD33.CAR and CD33.CAR(2A)CXCR4. The monocistronic CD33.CAR was used as control. We observed that both CD33.CAR(2A)CXCR4-CIKs (n=22, P<0.0001) and CXCR4(IRES)CD33.CAR-CIKs (n=9, P<0.0001) maintained CXCR4 over-expression during culture, whereas in CD33.CAR-CIKs was drastically downregulated (n=22). However, CD33.CAR expression was lower in CXCR4(IRES)CD33.CAR-CIKs (n=8, P<0.0001) compared to CD33.CAR-CIKs, while CD33.CAR(2A)CXCR4-CIKs (n=11) exhibited a significant co-expression of both proteins against control (P=0.001). CXCR4(IRES)CD33.CAR-CIKs and CD33.CAR(2A)CXCR4-CIKs maintained all CAR-associated in vitro effector functions, eliminating CD33+ KG1 target cell line, releasing cytokines (IL-2 and IFN-γ) and proliferating in an antigen-specific manner. However, CXCR4(IRES)CD33.CAR-CIKs exhibited lower effector responses against control, due to lower CAR expression. Chemotaxis assays toward recombinant CXCL12 confirmed both CXCR4(IRES)CD33.CAR-CIKs (n=7, P=0.01) and CD33.CAR(2A)CXCR4-CIKs (n=8, P=0.0006) displayed a migration advantage over CD33.CAR-CIKs (n=12) with a mean percentage of migration of 58.5% and 67.2% respectively, compared to 40.1%. Interestingly, CD33.CAR(2A)CXCR4-CIKs (n=2) showed an increased specific chemotactic response toward healthy (n=3) and AML-derived MSC (n=2) supernatants, which could be inhibited by the use of the CXCR4 antagonist Plerixafor. Moreover, when infused intravenously into NSG mice, significantly higher proportions of CD33.CAR(2A)CXCR4-CIKs were recovered in the femur BM compared to controls (P=0.0068). In conclusion, CD33.CAR(2A)CXCR4-CIKs, reaching the medullary niche more effectively, have the potential to more efficiently target the residing LSC responsible for the high relapse rates in AML. Disclosures Dotti: Tessa Therapeutics: Consultancy; Bellicum Pharmaceuticals: Consultancy; Catamaran: Consultancy. Biondi: Bluebird: Other: Advisory Board; Amgen: Honoraria; Incyte: Consultancy, Other: Advisory Board; Novartis: Honoraria; Colmmune: Honoraria.

Non-Viral Sleeping Beauty Transposon Engineered CD19-CAR-NK Cells Show a Safe Genomic Integration Profile and High Antileukemic Efficiency

Background: Natural Killer (NK) cells are known for their high intrinsic cytotoxic capacity. Recently, we and others showed that virally transduced NK cells equipped with a synthetic chimeric antigen receptor (CAR) targeting CD19 induced enhanced killing of acute lymphoblastic leukemia (ALL) cells. Here, we demonstrate for the first time that primary NK cells can be engineered using the non-viral Sleeping Beauty (SB) transposon/transposase system to stably express a CD19-CAR with a safe genomic integration profile and high anti-leukemic efficiency in vitro and in vivo. Methods: Primary NK cells were isolated from PBMCs from healthy donors. SB transposons vectorized as minicircles (MC), which encode either a Venus fluorescent protein or a CD19-CAR together with truncated EGFR (tEGFR) as a marker, were introduced in combination with the hyperactive SB100X transposase into primary NK cells via nucleofection. The genetically engineered NK cells were expanded using IL-15 cytokine stimulation under feeder-cell free conditions. Vector integration sites were mapped by analyzing the genomic region around each insertion site in genomic DNA from long-term cultivated gene-modified NK cells, engineered ether by lentiviral (LV) or SB-based technology. Stable gene delivery and biological activity were monitored by flow cytometry and cytotoxicity of CD19-CAR NK cells against CD19-positive ALL and CD19-negative cell lines. Results: Applying a protocol optimized with respect to nucleofection pulses, time points and plasmid ratios, primary NK cells showed long-lasting Venus expression (up to 50%) upon SB-mediated gene delivery with similar viability as non-treated (NT) NK cells during feeder-cell free ex-vivo expansion using IL-15. Likewise, SB transposon-engineered CD19-CAR NK cells displayed high viability, durable transgene expression (Fig 1 A), and no significant change in the NK cell phenotype profile. Next, we assessed vector integration into genomic safe harbors (GSH). GSH are defined as regions of human chromosomes that fulfill the following five criteria: not ultraconserved, >300 kb away from miRNA genes, >50 kb away from transcriptional start sites (TSS), >300 kb away from genes involved in cancer and outside transcription units. CD19-CAR NK cells generated using SB100X showed a significantly higher frequency of vector integration into GSH compared to LV-transduced CAR-NK cells and a significantly more-close to random nucleotide frequency (computer-generated random positions in the genome map to GSHs; random 43.68%; LV 14.78%, SB100X 23.99%; p<0.05) (Fig 1 B). MC.CD19-CAR NK cells generated with the SB platform demonstrated significantly higher cytotoxicity compared to NT NK cells against CD19-positive Sup-B15 ALL cells after long-term cultivation for two to three weeks and no loss of natural intrinsic cytotoxicity. After 4-hour co-culture, significantly enhanced specific tumor cell lysis was found for MC.CD19-CAR NK cells vs NT NK cells at all effector to target cell ratios (E:T) tested (E:T 20:1 83.88% vs 43.13%; E:T 10:1 75.18% vs 31.32%; E:T 5:1 67.38 vs 32.22%; E:T 1:1 42.54 vs 10.19%; p<0.05) (Fig 1 C). With regard to intrinsic natural cytotoxicity of NK cells, no significant decrease in cell killing was overserved for SB-gene-modified CD19-CAR NK cells compared to NT NK cells against CD19-negative K562 cells (E:T 5:1 83%; p<0.05) (Fig 1 D). Significantly enhanced antitumor potential of SB-generated CD19-CAR NK cells was confirmed in a systemic CD19-positive lymphoma xenograft model (NSG-Nalm-6/Luc) in vivo. After injection of 0.5x10 6 tumor cells per mouse and lymphoma engraftment, animals were treated with a single dose of 10x10 6 SB-modified CD19-CAR NK cells pooled from three different donors with a mean tEGFR/CAR expression of 34%. MC.CD19-CAR NK cell therapy resulted in rapid lymphoma eradication in all treated mice (n=4; p<0.05), whereas mice receiving similar amounts of NT NK cells showed progressive lymphoma growth comparable to untreated control mice (Fig 1 E-F). Conclusion: Taken together, the Sleeping Beauty transposon system represents an innovative gene therapy approach for non-viral engineering of safe, highly functional and relatively cost-efficient CAR-NK cells that may not only be suitable for ALL therapy but also for a broad range of other applications in cancer therapy. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.