Sleeping Beauty Research Articles

A Novel Hyperactive Variant of the Sleeping Beauty Transposase Facilitates Non-Viral Genome Engineering

The Sleeping Beauty (SB) transposon system is a useful tool for genetic applications, including gene therapy. We discovered a hyperactive variant of the SB100X transposase, called SB200X. This mutant, resulting from a specific amino acid replacement (Q124C), showed a ∼2-fold increase in transposition activity in various human and murine cells. Other amino acid replacements in position 124 also led to a hyperactive phenotype. Position 124 is located at the very edge of the linker region that connects the DNA-binding and catalytic domains of the transposase. Consistent with a role of the linker in an autoregulatory mechanism called overproduction inhibition (OPI) in the monophyletic group of mariner transposases, we show that the hyperactivity of Q124C manifests at high concentrations of the transposase, suggesting a partial resistance of SB200X to OPI. We demonstrate that the hyperactive phenotype of Q124C can be combined with features of other useful mutations in the SB transposase. Namely, Q124C improves the transposition efficiency of the previously described K248R variant, while maintaining or even slightly improving its safer genome-wide integration profile. SB200X transposase could enhance the utility of SB transposon-mediated genome engineering in preclinical and clinical applications.

Evaluation of Recombinant Antibody Production Efficiency in CHO Cells with Sleeping Beauty Transposon Vector System

Chinese hamster ovary (CHO) mammalian cell lines are widely used as cell platforms in biopharmaceutical productions. Different transfection systems are employed for the integration of the target gene cassette into the cell genome and have limitations, such as (i) the integration region in the genome, (ii) the size of the target cassette, and (iii) long selection periods for stable expression. Transposon systems can be utilized to overcome the limitations mentioned in the efficient production of commercially significant recombinant proteins. This study aims to demonstrate the differences in production potential and selection periods by using a specially designed vector system for random genome integration in CHODG44 DHFR -/- cells and the Sleeping Beauty (SB) transposon system. In this context, the optimal transfer ratio between the donor and the helper plasmid was determined for the most efficient co-transfection in the SB transposon system. According to the results, the pools obtained using the SB transposon system had titers ranging from 1300 to 2600 mg/L in 13-day fed-batch studies, while the pool obtained using the random transfer system had a titer of 0.056 mg/L. Additionally, stable cell pools obtained using the transposon system underwent selection in a short period of 52 days, compared to over 100 days for the pool obtained through random transfer. Considering all these results together, it is demonstrated that stable CHO pools obtained using the optimal SB transposon system can achieve high-efficiency monoclonal antibody production in a short period, making it an optimal production platform in the biopharmaceutical field.

Stable transduction of the neonatal mouse liver using a hybrid rAAV/sleeping beauty transposon gene delivery system.

Conventional adeno-associated viral (AAV) vectors, while highly effective in quiescent cells such as hepatocytes in the adult liver, confer less durable transgene expression in proliferating cells owing to episome loss. Sustained therapeutic success is therefore less likely in liver disorders requiring early intervention. We have previously developed a hybrid, dual virion approach, recombinant AAV (rAAV)/piggyBac transposon system capable of achieving stable gene transfer in proliferating hepatocytes at levels many fold above conventional AAV vectors. An alternative transposon system, Sleeping Beauty, has been widely used for ex vivo gene delivery; however liver-targeted delivery using a hybrid rAAV/Sleeping Beauty approach remains relatively unexplored. We investigated the capacity of a Sleeping Beauty (SB)-based dual rAAV virion approach to achieve stable and efficient gene transfer to the newborn murine liver using transposable therapeutic cassettes encoding coagulation factor IX or ornithine transcarbamylase (OTC). At equivalent doses, rAAV/SB100X transduced hepatocytes with high efficiency, achieving stable expression into adulthood. Compared with conventional AAV, the proportion of hepatocytes transduced, and factor IX and OTC activity levels, were both markedly increased. The proportion of hepatocytes stably transduced increased 4- to 8-fold from <5%, and activity levels increased correspondingly, with markedly increased survival and stable urinary orotate levels in the OTC-deficient Spfash mouse following elimination of residual endogenous murine OTC. The present study demonstrates the first in vivo utility of a hybrid rAAV/SB100X transposon system to achieve stable long-term therapeutic gene expression following delivery to the highly proliferative newborn mouse liver. These results have relevance to the treatment of genetic metabolic liver diseases with neonatal onset.

In vivo AAV-SB-CRISPR screens of tumor-infiltrating primary NK cells identify genetic checkpoints of CAR-NK therapy.

Natural killer (NK) cells have clinical potential against cancer; however, multiple limitations hinder the success of NK cell therapy. Here, we performed unbiased functional mapping of tumor-infiltrating NK (TINK) cells using in vivo adeno-associated virus (AAV)-SB (Sleeping Beauty)-CRISPR (clustered regularly interspaced short palindromic repeats) screens in four solid tumor mouse models. In parallel, we characterized single-cell transcriptomic landscapes of TINK cells, which identified previously unexplored subpopulations of NK cells and differentially expressed TINK genes. As a convergent hit, CALHM2-knockout (KO) NK cells showed enhanced cytotoxicity and tumor infiltration in mouse primary NK cells and human chimeric antigen receptor (CAR)-NK cells. CALHM2 mRNA reversed the CALHM2-KO phenotype. CALHM2 KO in human primary NK cells enhanced their cytotoxicity, degranulation and cytokine production. Transcriptomics profiling revealed CALHM2-KO-altered genes and pathways in both baseline and stimulated conditions. In a solid tumor model resistant to unmodified CAR-NK cells, CALHM2-KO CAR-NK cells showed potent in vivo antitumor efficacy. These data identify endogenous genetic checkpoints that naturally limit NK cell function and demonstrate the use of CALHM2 KO for engineering enhanced NK cell-based immunotherapies.

Overcoming recognition delays in disruptive research: The impact of team size, familiarity, and reputation

The relationship between disruption and delayed recognition is a critical research topic, yet the connection between the degree of disruption and delayed acknowledgment remains unclear. This study investigates the extent of recognition delay for disruptive papers using the SciSciNet dataset. We conducted a quantitative analysis based on this extensive dataset to examine the relationship between the Disruption Index and the Sleeping Beauty Index, revealing that highly disruptive papers often face a latency period before gaining acknowledgment, with significant variations across disciplines and over time. Our analysis of team dynamics indicates that larger teams, the presence of high-impact authors, fixed teams, and hierarchically structured teams can significantly reduce this delay. These findings provide insights into optimizing team strategies and understanding the complexities of academic recognition. They offer valuable implications for researchers and policymakers aiming to foster and accelerate the acknowledgment of groundbreaking scientific contributions.

UBXN1 promotes liver tumorigenesis by regulating mitochondrial homeostasis

BackgroundThe maintenance of mitochondrial homeostasis is critical for tumor initiation and malignant progression because it increases tumor cell survival and growth. The molecular events controlling mitochondrial integrity that facilitate the development of hepatocellular carcinoma (HCC) remain unclear. Here, we report that UBX domain-containing protein 1 (UBXN1) hyperactivation is essential for mitochondrial homeostasis and liver tumorigenesis.MethodsOncogene-induced mouse liver tumor models were generated with the Sleeping Beauty (SB) transposon delivery system. Assessment of HCC cell growth in vivo and in vitro, including tumour formation, colony formation, TUNEL and FACS assays, was conducted to determine the effects of UBXN1 on HCC cells, as well as the involvement of the UBXN1-prohibitin (PHB) interaction in mitochondrial function. Coimmunoprecipitation (Co-IP) was used to assess the interaction between UBXN1 and PHB. Liver hepatocellular carcinoma (LIHC) datasets and HCC patient samples were used to assess the expression of UBXN1.ResultsUBXN1 expression is commonly upregulated in human HCCs and mouse liver tumors and is associated with poor overall survival in HCC patients. UBXN1 facilitates the growth of human HCC cells and promotes mouse liver tumorigenesis driven by the NRas/c-Myc or c-Myc/shp53 combination. UBXN1 interacts with the inner mitochondrial membrane protein PHB and sustains PHB expression. UBXN1 inhibition triggers mitochondrial damage and liver tumor cell apoptosis.ConclusionsUBXN1 interacts with PHB and promotes mitochondrial homeostasis during liver tumorigenesis.

Establishment of primary prostate epithelial and tumorigenic cell lines using a non-viral immortalization approach

BackgroundResearch on prostate cancer is mostly performed using cell lines derived from metastatic disease, not reflecting stages of tumor initiation or early progression. Establishment of cancer cell lines derived from the primary tumor site has not been described so far. By definition, cancer cells are able to be cultured indefinitely, whereas normal epithelial cells undergo senescence in vitro. Epithelial cells can be immortalized, accomplished by using viral integration of immortalization factors. Viral approaches, however, might be impaired by regulatory and safety issues as well as random integration into regulatory genetic elements, modifying precise gene expression. We intend to use surgical specimen of prostate cancer patients to (i) prove for establishment of cancer cell lines, and (ii) perform non-viral, Sleeping Beauty (SB) transposase-based immortalization of prostate epithelial cells.MethodsRadical prostatectomy samples of prostate cancer patients (n = 4) were dissociated and cultured in vitro. Cells were cultivated either without or after non-viral, Sleeping-Beauty transposase-based stable transfection with immortalization factors SV40LT and hTERT. Established cell lines were analyzed in vitro and in vivo for characteristics of prostate (cancer) cells.ResultsInitial cell cultures without genetic manipulation underwent senescence within ≤ 15 passages, demonstrating inability to successfully derive primary prostate cancer cell lines. By using SB transposase-based integration of immortalization factors, we were able to establish primary prostate cell lines. Three out of four cell lines displayed epithelial characteristics, however without expression of prostate (cancer) characteristics, e.g., androgen receptor. In vivo, one cell line exhibited tumorigenic potential, yet characteristics of prostate adenocarcinoma were absent.ConclusionWhereas no primary prostate cancer cell line could be established, we provide for the first-time immortalization of primary prostate cells using the SB transposase system, thereby preventing regulatory and molecular issues based on viral immortalization approaches. Although, none of the newly derived cell lines demonstrated prostate cancer characteristics, tumor formation was observed in one cell line. Given the non-prostate adenocarcinoma properties of the tumor, cells have presumably undergone oncogenic transformation rather than prostate cancer differentiation. Still, these cell lines might be used as a tool for research on prostate cancer initiation and early cancer progression.

Development of a compact bidirectional promoter-driven dual chimeric antigen receptor (CAR) construct targeting CD19 and CD20 in the Sleeping Beauty (SB) transposon system

BackgroundA bidirectional promoter-driven chimeric antigen receptor (CAR) cassette provides the simultaneous expression of two CARs, which significantly enhances dual antigen-targeted CAR T-cell therapy.MethodsWe developed a second-generation CAR directing CD19 and...

Abstract 1998: ABCB1 promotes uveal melanoma cell resistance to FR900359 and identifies a tumor cell subpopulation with a distinct gene expression signature

Abstract Uveal melanoma (UM) is the leading form of intraocular cancer in adults and the second most common form of melanoma. Although rare, UM has a median survival rate of ~1 year after metastasis occurs. Metastatic UM is largely refractory to treatment and there are no effective pharmacological therapies, resulting in poor overall survival. In &amp;gt;90% UM cases, the oncogenic initiator proteins are constitutively active mutant GNAQ or GNA11 proteins (GNAQ/11). FR900359 (FR) is a natural compound that inhibits GNAQ/11 and has anti-proliferative effects on UM cells. To identify key oncogenic mechanisms downstream of mutant GNAQ/11, we performed a Sleeping Beauty (SB) transposon forward genetic screen to identify drivers of FR resistance. The SB screen identified ATP-binding cassette sub-family B member 1 (ABCB1) as one of the top genes upregulated by SB in FR-resistant UM cell populations. We determined that although ABCB1 does not provide immediate resistance to FR, the overexpression of ABCB1 leads to emergence of resistant colonies at a much higher rate than spontaneous resistance, validating ABCB1 as contributors to FR resistance. Unexpectedly, immunofluorescence revealed that a relatively small fraction of UM cells strongly expresses the transgenes after stable transfection. Immunoblotting confirmed that ABCB1 expression was initially modest in ABCB1-transfected populations but increased in the cells that developed resistance to FR. To examine the kinetics of FR resistance, population doubling versus time (PDVT) assays were performed in UM cells expressing ABCB1 or empty vector control treated with FR or vehicle. FR potently suppressed the proliferation of vector control cells. Drug-naïve ABCB1-transfected cells developed resistance to FR over time but accomplished fewer population doublings than vehicle-treated cells in the same time interval. Once transgenic populations emerged as resistant populations in FR, they grew similarly in drug or vehicle. Ongoing RNA-sequencing analysis from PDVT samples has identified distinct gene expression signatures associated with FR treatment and ABCB1 expression. These findings are expected to illuminate tumor cell heterogeneity amongst UM cell populations that may be related to drug resistance and metastasis. Citation Format: Sarina Dion Murray, Jesse D. Riordan, Eric R. Anderson, Michael D. Onken, Kendall J. Blumer, Adam J. Dupuy, Christopher S. Stipp. ABCB1 promotes uveal melanoma cell resistance to FR900359 and identifies a tumor cell subpopulation with a distinct gene expression signature [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1998.

Abstract 1632: Dissecting AMBRA1-cyclin D axis deregulation in embryonal tumors of the central nervous system

Abstract Dissecting AMBRA1-cyclin D axis deregulation in embryonal tumors of the centralnervous systemEmbryonal tumors of the central nervous system (CNS) are malignant growths originating from embryonic neural stem cells, primarily affecting pediatric patients. These tumors pose a significant threat to children's health, leading to high morbidity and mortality rates. To improve survival rates and reduce morbidity, there is a critical need for advanced molecular diagnostics and innovative therapies. Among these tumors are medulloblastomas (MB) and other embryonal tumors, previously classified as primitive neuroectodermal tumors (CNS-PNET). These malignancies are characterized by uncontrolled cell proliferation, primarily driven by abnormalities in cell cycle proteins. Consequently, targeting cell cycle regulators has emerged as a promising approach for their treatment. Two key signaling pathways, namely the MYC and the cyclin D-CDK-RB pathway, govern the decision-making process related to genome replication. Mutations in both MYC and the CDK4/6/cyclin D pathway, leading to the functional inactivation of RB, have been identified as relevant factors in the development of CNS-PNET and medulloblastomas. Our seminal research has highlighted the crucial role of AMBRA1, an upstream master regulator orchestrating the transition from G1 to S phase (Maiani &amp; Milletti et al., 2021). Specifically, AMBRA1 modulates the abundance of D-type cyclins and MYC by facilitating their proteasomal degradation, thereby ensuring genomic stability during DNA replication.Based on these discoveries, we propose that AMBRA1 functions as a multifaceted tumor suppressor across various cancer types. An unbiased Sleeping Beauty analysis identified AMBRA1 as a genetic driver of CNS-PNET, providing a strong foundation for investigating its role further (Beckmann et al., 2019). We hypothesize that the simultaneous deletion of AMBRA1 and p53 in an inducible knockout mouse model can lead to the spontaneous development of CNS-PNET or medulloblastoma, contingent on the specific targeted cell lineage. Additionally, our research demonstrates that AMBRA1 deficiency confers resistance to CDK4/6 inhibitors while creating a synthetic lethality with CHK1 inhibitors. Consequently, we propose that a comparative analysis of AMBRA1, D-type cyclins, and Myc family expression levels could serve as a diagnostic marker. This marker could facilitate patient-based stratification for CHK1 inhibitor treatment efficacy, enabling the differentiation of non- responsive cases to CDK4/6 chemotherapy. Citation Format: Giacomo Milletti, Giulia Cadeddu, Alba Adelantado Rubio, Caterina Ferraina, Apolinar Maya-Mendoza, Francesco Cecconi. Dissecting AMBRA1-cyclin D axis deregulation in embryonal tumors of the central nervous system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1632.

Abstract PR-011: The role of tumor microenvironment in metastasis and relapses of medulloblastoma using immunocompetent mouse models

Abstract Medulloblastoma (MB) is an embryonal malignant tumor that arises in the cerebellum of infants, children, and adolescents. Despite highly aggressive therapies, including surgery, radiation, and chemotherapy, metastasis remains the primary cause of death in these patients. The tumor microenvironment (TME) has an important role in metastasis and relapse in several cancer types, but it’s role in high-risk MBs (metastatic and relapse) has been understudied. Here, we investigated the process of relapse in an immunocompetent Shh MB mouse model based on somatic mutagenesis induced by Sleeping Beauty (SB)-mediated transposition on a genetically engineered strain carrying a loss-of-function Patched 1 gene (Ptch1) mutation. Lack of one functional copy of the Ptch1 gene predisposes this strain to develop MB. Somatic mutations induced by the transposon increase penetrance of tumor formation in the triple mutant mice carrying the SB transposon system and the Ptch1 mutation. We used this model to study the mechanisms of chemoresistance to cisplatin and cyclophosphamide, that constitute the backbone of the high-dose chemotherapy used to treat patients under the age of 3, who cannot receive craniospinal irradiation. By treating the SB mouse model with surgery followed by high-dose fractionated chemotherapy, we developed a model of MB chemoresistance which is relevant to the infant SHH-MB patient population. While chemotherapy improved mouse survival, only a small number of animals were cured, mirroring the outcome seen in infant patients with Shh MB. Deep sequencing and mapping of transposon-gDNA junctions, along with gene-centric common insertion site analysis (gCIS), revealed that Trp53 transposon insertions are found exclusively at relapse. In the metastatic compartment we observed convergence of gCISes on genes coding for epigenetic regulators, among them a prominent enrichment was found for PRC2 complex genes. RNA-seq analysis on the transcriptome of primary and recurrent tumors indicated that a majority of the differentially expressed genes are associated with inflammatory responses, IL6, and TNF-α/NF-κB signaling. Immunohistochemistry to visualize the expression levels of inflammatory markers: IL-6, CCL2, and F4-80 and spatial targeted multiplexed assays (Geomx) confirmed the increase of immune cells at progression. ChIP-qPCR on cytokine loci revealed binding of PRC2 and deposition of PRC2 mediated repressive histone mark H3K27me3 on the promoter of CCL2 highlighting LOF mutations in PRC2 components as potential epigenetic mechanisms for immune recruitment at progression. Inhibition of immune activation through pharmacological strategies might be a possible therapeutic option for infant SHH-MB. Citation Format: Niusha Khazaei, Ana Castillo Orozco, Wajih Jawhar, Geoffroy Danieau, Kiran Narta, Michael Taylor, Sorana Morrissy, Ana Stuecklin, Livia Garzia. The role of tumor microenvironment in metastasis and relapses of medulloblastoma using immunocompetent mouse models [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr PR-011.

The Theme Of Virginity In Japanese Literature -Example of ‘The House of the Sleeping Beauties-

Birçok toplumda olduğu gibi, Japon toplumunda da tabu olarak görülen bekâret, II. Dünya Savaşı sonrası Japonya’sında kadının sosyal statüsünün değişmesiyle birlikte önemini kaybetmeye başlamıştır. Kadın ve bekâret konuları Nobel ödüllü Japon yazar Kawabata Yasunari’nin eserlerinde de çokça görülmektedir. Kawabata, eserlerinde kadınların görsel tasvirlerini büyük titizlikle yapmakta, kadınların toplum içi rollerini vurgulamakta ve aynı zamanda bekâret temasını da sıkça işlemektedir. Bu eserlerden birisi de Uykuda Sevilen Kızlar (『眠れる美女』 - Nemureru Bijo) eseridir. Bu eser, II. Dünya Savaşı sonrasında yayınlanan ve bakire kızlar ile birlikte uyuyan, yaşlı Eguchi’yi konu almaktadır.&#x0D; Bu çalışmada, Kawabata’nın savaş sonrası eseri Uykuda Sevilen Kızlar ele alınmış olup, bakire kızlarla birlikte uyuyan Eguchi karakterinin “bekâret” üzerine düşünceleri, bu düşüncelerdeki değişim, Todorov’un nitelemeleri ve değişimi ön plana çıkaran yapısal anlatı çözümleme yöntemi ile ortaya konulmuştur. Böylece Japon toplumundaki savaş sonrası toplumsal bir tabu olan “bekâret” olgusuna yönelik fikir değişimi ile Kawabata’nın eseri arasındaki paralellik ortaya konulmuş, Japon toplumundaki bir algı değişimine tanıklık edilmiştir.

Suppression of Sleeping Beauty-induced Gliomagenicity in Ts1Cje Mice, a Model of Down Syndrome.

Individuals with Down syndrome (DS), attributed to triplication of human chromosome 21 (Hsa21), exhibit a reduced incidence of solid tumors. However, the prevalence of glioblastoma among individuals with DS remains a contentious issue in epidemiological studies. Therefore, this study examined the gliomagenicity in Ts1Cje mice, a murine model of DS. We employed the Sleeping Beauty transposon system for the integration of human oncogenes into cells of the subventricular zone of neonatal mice. Notably, Sleeping Beauty-mediated de novo murine gliomagenesis was significantly suppressed in Ts1Cje mice compared to wild-type mice. In glioblastomas of Ts1je mice, we observed an augmented presence of M1-polarized tumor-associated macrophages and microglia, known for their anti-tumor efficacy in the early stage of tumor development. Our findings in a mouse model of DS offer novel perspectives on the diminished gliomagenicity observed in individuals with DS.

Sleeping Beauty transposon mutagenesis in mouse intestinal organoids identifies genes involved in tumor progression and metastasis.

To identify genes important for colorectal cancer (CRC) development and metastasis, we established a new metastatic mouse organoid model using Sleeping Beauty (SB) transposon mutagenesis. Intestinal organoids derived from mice carrying actively mobilizing SB transposons, an activating KrasG12D, and an inactivating ApcΔ716 allele, were transplanted to immunodeficient mice. While 66.7% of mice developed primary tumors, 7.6% also developed metastatic tumors. Analysis of SB insertion sites in tumors identified numerous candidate cancer genes (CCGs) identified previously in intestinal SB screens performed in vivo, in addition to new CCGs, such as Slit2 and Atxn1. Metastatic tumors from the same mouse were clonally related to each other and to primary tumors, as evidenced by the transposon insertion site. To provide functional validation, we knocked out Slit2, Atxn1, and Cdkn2a in mouse tumor organoids and transplanted to mice. Tumor development was promoted when these gene were knocked out, demonstrating that these are potent tumor suppressors. Cdkn2a knockout cells also metastasized to the liver in 100% of the mice, demonstrating that Cdkn2a loss confers metastatic ability. Our organoid model thus provides a new approach that can be used to understand the evolutionary forces driving CRC metastasis and a rich resource to uncover CCGs promoting CRC.

Engineering a CRISPRoff Platform to Modulate Expression of Myeloid Cell Leukemia (MCL-1) in Committed Oligodendrocyte Neural Precursor Cells.

In vitro differentiation of human pluripotent stem cell (hPSC) model systems has furthered our understanding of human development. Techniques used to elucidate gene function during early development have encountered technical challenges, especially when targeting embryonic lethal genes. The introduction of CRISPRoff by Nuñez and collaborators provides an opportunity to heritably silence genes during long-term differentiation. We modified CRISPRoff and sgRNA Sleeping Beauty transposon vectors that depend on tetracycline-controlled transcriptional activation to silence the expression of embryonic lethal genes at different stages of differentiation in a stable manner. We provide instructions on how to generate sgRNA transposon vectors that can be used in combination with our CRISPRoff transposon vector and a stable hPSC line. We validate the use of this tool by silencing MCL-1, an anti-apoptotic protein, which results in pre-implantation embryonic lethality in mice; this protein is necessary for oligodendrocyte and hematopoietic stem cell development and is required for the in vitro survival of hPSCs. In this protocol, we use an adapted version of the differentiation protocol published by Douvaras and Fossati (2015) to generate oligodendrocyte lineage cells from human embryonic stem cells (hESCs). After introduction of the CRISPRoff and sgRNAs transposon vectors in hESCs, we silence MCL-1 in committed oligodendrocyte neural precursor cells and describe methods to measure its expression. With the methods described here, users can design sgRNA transposon vectors targeting MCL-1 or other essential genes of interest to study human oligodendrocyte development or other differentiation protocols that use hPSC model systems. Key features • Generation of an inducible CRISPRoff Sleeping Beauty transposon system. • Experiments performed in vitro for generation of inducible CRISPRoff pluripotent stem cell line amenable to oligodendrocyte differentiation. • Strategy to downregulate an essential gene at different stages of oligodendrocyte development.

RBFOX2 deregulation promotes pancreatic cancer progression and metastasis through alternative splicing

RNA splicing is an important biological process associated with cancer initiation and progression. However, the contribution of alternative splicing to pancreatic cancer (PDAC) development is not well understood. Here, we identify an enrichment of RNA binding proteins (RBPs) involved in splicing regulation linked to PDAC progression from a forward genetic screen using Sleeping Beauty insertional mutagenesis in a mouse model of pancreatic cancer. We demonstrate downregulation of RBFOX2, an RBP of the FOX family, promotes pancreatic cancer progression and liver metastasis. Specifically, we show RBFOX2 regulates exon splicing events in transcripts encoding proteins involved in cytoskeletal remodeling programs. These exons are differentially spliced in PDAC patients, with enhanced exon skipping in the classical subtype for several RBFOX2 targets. RBFOX2 mediated splicing of ABI1, encoding the Abelson-interactor 1 adapter protein, controls the abundance and localization of ABI1 protein isoforms in pancreatic cancer cells and promotes the relocalization of ABI1 from the cytoplasm to the periphery of migrating cells. Using splice-switching antisense oligonucleotides (AONs) we demonstrate the ABI1 ∆Ex9 isoform enhances cell migration. Together, our data identify a role for RBFOX2 in promoting PDAC progression through alternative splicing regulation.

Abstract B013: Investigating the role of LINE-1 ORF1 protein in a syngeneic mouse melanoma model

Abstract Long interspersed element type 1 (LINE-1) is a class of transposable elements that make up 17% of human genome. Somatic LINE-1 insertions promote genomic instability and have been implicated in the evolution of various human cancer types. A full-length LINE-1 has two open reading frames (ORF1 and ORF2) that encode proteins essential for its replication in the genome. It has been well documented that LINE-1 ORF1 protein (ORF1p) is overexpressed in tumor tissues as compared to non-tumor tissues. However, whether ORF1p itself is oncogenic remains controversial. Downregulation of ORF1p has been shown to reduce cancer cell proliferation and metastasis. ORF1p has also been associated with the modulation of tumor microenvironment and activation of oncogenic pathways by interacting with cellular proteins. We are interested in the role of LINE-1 ORF1p in a syngeneic mouse melanoma model. B16-F10 is a mouse melanoma cell line derived from a C57BL/6 mouse. It had an undetectable level of ORF1p by Western blot. To investigate the effect of ORF1p overexpression in vitro and in vivo, we started with establishing ORF1p expression in B16-F10 cells. We utilized the Sleeping Beauty gene delivery system to make single-cell clones expressing the protein. Cell proliferation and migration assays give insight into the effect of ORF1p overexpression in vitro. Growth of the parental and ORF1p-expressing B16-F10 cells in C57BL/6 mice informs the effect of ORF1p overexpression in vivo. This study represents an important first step to establishing the role of ORF1p in melanoma, a common cancer worldwide. Citation Format: Raj Nandani, Wenfeng An. Investigating the role of LINE-1 ORF1 protein in a syngeneic mouse melanoma model [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B013.

Forward Genetic Screen Implicates Drivers of Leukemic Progression in a Novel Model of Trp53 R270H myelodysplastic Syndrome

Myelodysplastic syndrome (MDS) is characterized by bone marrow failure and a highly variable clinical course. The most catastrophic complication of MDS is transformation to secondary acute myeloid leukemia (sAML). Notably, mutations in TP53 confer the single highest risk of transformation to sAML and death. However, some patients with TP53 mutated MDS do not develop sAML, suggesting that additional genetic events cooperate with TP53 mutations to transform MDS to sAML. Understanding the mechanisms of transformation of MDS to sAML could provide targets for therapeutic intervention. To model the genetics of MDS, we crossed mice bearing Trp53 R270H ( Trp53 is the murine TP53 gene) and deletion of genes syntenic with human chromosome 5q (del(5q)). To discover how additional mutations contribute to disease progression, we utilized Sleeping Beauty (SB) transposon mutagenesis in Trp53 R270H/del(5q) mice. SB transposase mobilized SB mutagenic T2/Onc transposons which randomly insert within the genome. T2/Onc transposons are designed to induce gain or loss of function alterations depending on the site and orientation of insertion with respect to targeted genes. We used the Mx1-Cre transgene to activate SB transposase and T2/Onc transposition in hematopoietic progenitors. Trp53 R270H anddel(5q)(or cytogenetically normal, CN) mice were crossed to SB mice to generate donor mice of the following genotypes: Trp53 R270H/del(5q)/ SB, Trp53 R270H/CN /SB, Trp53 WT/del(5q)/ SB, Trp53 WT/CN/ SB mice, and mice without SB transposition, (no transposition, NT: Trp53 R270H/del(5q) /NT). Bone marrow cells were transplanted into recipients, and SB insertional mutagenesis was activated using pI-pC to activate Cre. Mice receiving Trp53 WT/CN/ SB bone marrow developed more frequent T-cell leukemia (n=3/10) than myeloid leukemia (n=1/10). In contrast, mice receiving Trp53 R270H/del(5q)/SB and Trp53 R270H/CN /SB bone marrow developed predominantly myeloid leukemia (n=14/28) more commonly than T-cell leukemia (1/28). Mixed phenotype leukemia was seen in 7/28 of these mice. Together, these data demonstrate a strong bias towards myeloid disease in SB-mutagenized Trp53 R270H bone marrow. To identify genes with SB insertions, we performed RNA sequencing to detect SB T2/Onc transposon-endogenous genefusion transcripts. Among Trp53 WT/CN/ SB leukemias, the most common recurrent SB fusions involved Notch1 and Ikzf1 as has previously reported for SB-associated T-cell leukemias . Among Trp53 R270H/del(5q)/SB and Trp53 R270H/CN /SB leukemias, the most common recurrent SB-fusions involved Erg, Eras and Il2rb with Erg fusions detected 85% of Trp53 R270H leukemias (n=17/20). SB inserted upstream of Erg promoter indicating that these fusions likely upregulate expression of Erg. Indeed, Erg levels are significantly higher in leukemias that express SB-Erg fusions relative to leukemias that do not (p&amp;lt;0.0023). ERG is not recurrently mutated in human AML, but the ERG gene locus is commonly amplified, especially TP53 mutant AML. ERG is known to support normal hematopoietic stem cell self-renewal. Notably, Erg-insertions were also detected in a model of MDS expressing stabilized cyclin E with SB-mediated progression to erythroleukemia (Loeb 2019). Using gene set enrichment analysis, we found that hematopoietic stem cell and leukemic stem cell signatures are enriched in Erg-SB fusion leukemias. In our analyses of two independent data sets (TCGA and BEAT AML), stem cell signatures are also among the most highly enriched pathways in human AMLs expressing high ERG levels. Furthermore, in a human AML single cell RNA sequencing dataset (van Galen 2019), we found that ERG expression is highest in AML cells with the most immature stem and progenitor-like features. Together, these findings implicate a role for ERG as a driver of progression of MDS to AML by enhancing aberrant self-renewal. In summary, we present a novel murine model of Trp53/del(5q) MDS. In this model, Erg upregulation is associated with progression to AML and upregulation of leukemia stem cell gene expression profiles. These data implicate ERG as a major contributor to progression of MDS to secondary AML in the setting of mutant p53. Understanding the mechanisms of disease progression and self-renewal in myeloid malignancies with p53 mutations is critical to define effective therapeutic strategies in these rapidly fatal, treatment resistant diseases.

The Court and the Sleeping Beauty 2.0: Filling the Contractual Gap, or Making Valid Consumer Contracts to the Detriment of the Non-consumer?

Abstract This paper is a critical presentation of the CJEU case-law on UCTD, with particular reference to the non-filling of the contractual gap resulting from the unfairness test by applying ‘dispositive’ law or supplementary interpretation. The result contradicts the principles and methods of national civil law: not only does the balance in the contract suffer under the position the CJEU has chosen, as it creates alternated forms of balance of interests rather than the identical ones sought by the parties, but moreover, the increasing number of different cases imposes an ambiguous approach on the CJEU, since European judges have to ask on a case-by-case basis what the best interest of the consumer is. By adopting such a functional approach in favour of the substantive justice of the contract, the ECJ decided with questionable results with regard to national civil law, while methodological legal questions as to whether the contract should stand as it is, following the unfairness test, or whether a supplementation of the contract should take place are still open. It is therefore of critical importance to achieve an optimal combination between a fair balance of the contract and effective consumer protection.423

Manufacturing, Characterization and Anti-Tumor Activity of Allogeneic CD19-CAR Cytokine-Induced Killer (CIK) Cells Engineered with Non-Viral Sleeping Beauty Transposon System and Armored with IL-18

Rationale &amp; Objective: Adoptive cell therapy by chimeric antigen receptor- engineered cytokine-induced killer (CAR-CIK) cells has demonstrated high therapeutic potential with minimal alloreactivity and little to no toxicity in B-cell acute lymphoblastic leukemia patients, but further development is required to improve anti-tumor activity and persistence in vivo. The expression of the proinflammatory cytokine interleukin-18 (IL-18) has been shown to greatly enhance CAR-T cells potency and persistence. The purpose of the work described here was to develop a workflow for the manufacture of functional CAR-CIK cells, using the non-viral Sleeping Beauty transposon system, that co-express a second generation CD19-targeted CAR and the proinflammatory cytokine IL-18, hereafter referred to as CARCIK-1918 cells. The CAR molecule expressed by these cells includes an attenuated CD28 intracellular domain for enhancement of anti-tumor activity and in vivo persistence. Characterization of the manufactured armored CARCIK-1918 cells and evaluation of their anti-tumor activity in vitro and in vivo were performed. Methods: PBMCs isolated from healthy donors were cultured with IFN-γ, stimulated with anti-CD3 and IL-2, electroporated for non-viral genetic modification with Sleepy Beauty transposase RNA (SB100X) and pT4-1928'z-IL18 bicistronic plasmid DNA, and cultured for 15 days (17-day process). In process and final product characterization included monitoring of cell culture composition, cell expansion and viability, and evaluation of the expression of CAR, CIK proteins CD56 and NKG2D, and memory phenotype markers using flow cytometry. The final product CARCIK-1918 cells were further characterized by in vitro stimulation with CD19 + REH tumor cells to assess tumor killing activity and IL-18 secretion. CARCIK-1918 in vivo efficacy and persistence were also investigated in an NSG/Daudi mouse model. Mice were grafted with CD19 + Daudi tumor cells and, after 2 days, received an intravenous injection of 5x10 6 or 10x10 6 CARCIK-1918 cells. Daudi-grafted untreated mice were used as controls. Survival was monitored, and tumor burden and CAR-CIK cell persistence evaluated by using flow cytometry to assess human CD19 + and human CD3 + cells respectively, in peripheral blood, bone marrow, spleen, and kidney. Results: We manufactured &amp;gt;15 lots of CARCIK-1918 cells. After electroporation on day 2, CAR expression was already detected at day 7 and increased during the expansion culture. After the 17-day process, the range of harvested cells was 4.4E+09 to 1.6E+10 viable cells, with a cell viability &amp;gt;94%, and a fold expansion that varied among lots (range 20.1 - 161.4). The final CARCIK-1918 cells were predominantly CD3 +, expressed both the CAR (range 13 - 81%) and CIK-specific markers CD56 (range 27 - 88%) and NKG2D (range 59 - 86%) at variable levels among batches, and showed mostly a central memory/effector memory phenotype. CARCIK-1918 cells demonstrated potent and specific in vitro cytotoxicity towards the CD19 + REH target cell line. Cells secreted IL-18 only in the presence of CD19 + target cells (range 62.17 - 560.58 pg/mL). In addition, CARCIK-1918 cells had a substantial anti-leukemic activity in vivo. CARCIK-1918 injection resulted in a significant dose-dependent reduction of tumor growth and improved survival, as compared with untreated NSG/Daudi mice. CARCIK-1918 cells persisted in vivo in mouse peripheral blood up to 90 days. Post-mortem analysis demonstrated that CARCIK-1918 cells from both dose levels persisted over time and limited the level of leukemic cell dissemination in the animals, with 10x10 6 regimen allowing an almost total control of the disease progression in all tissue examined. Conclusion: In conclusion, this study demonstrates that manufacturing of armored CAR-CIK cells using Sleepy Beauty non-viral transposon system and 2 nd generation CD19 CAR with an attenuated CD28 costimulatory domain and armored with IL-18 is feasible and allows rapid and efficient expansion of highly potent CARCIK-1918 cells. These cells demonstrated potent and specific anti-tumor activity in vitro and a robust anti-leukemic activity in vivo. These findings provide the first reported successful production of IL-18 armored CAR-CIK cells offering a promising strategy for the treatment of B-cell malignancies.