Sleeping Beauty Transposon System Research Articles

Novel mechanisms of action of emerging therapies of hereditary thrombotic thrombocytopenic purpura.

Hereditary thrombotic thrombocytopenic purpura (hTTP) is caused by deficiency of plasma ADAMTS13 activity, resulting from ADAMTS13 mutations. ADAMTS13 cleaves ultra large von Willebrand factor (VWF), thus reducing its multimer sizes. Hereditary deficiency of plasma ADAMTS13 activity leads to the formation of excessive platelet-VWF aggregates in small arterioles and capillaries, resulting in hTTP. PubMed search from 1956-2024 using thrombotic thrombocytopenic purpura and therapy identified 3,675 articles. Only the articles relevant to the topic were selected for discussion, which focuses on pathophysiology, clinical presentations, and mechanisms of action of emerging therapeutics for hTTP. Current therapies include infusion of plasma, or coagulation factor VIII, or recombinant ADAMTS13. Emerging therapies include anti-VWF A1 aptamers or nanobody and gene therapies with adeno-associated viral vector or self-inactivated lentiviral vector or a Sleeping Beauty transposon system for a long-term expression of a functional ADAMTS13 enzyme. Frequent plasma infusion remains to be the standard of care in most parts of the world, while recombinant ADAMTS13 has become the treatment of choice for hTTP in some of the Western countries. The success of gene therapies in preclinical models may hold a promise for future development of these novel approaches for a cure of hTTP.

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 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.

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.

Molecular Mechanisms in Pathophysiology of Mucopolysaccharidosis and Prospects for Innovative Therapy.

Mucopolysaccharidoses (MPSs) are a group of inborn errors of the metabolism caused by a deficiency in the lysosomal enzymes required to break down molecules called glycosaminoglycans (GAGs). These GAGs accumulate over time in various tissues and disrupt multiple biological systems, including catabolism of other substances, autophagy, and mitochondrial function. These pathological changes ultimately increase oxidative stress and activate innate immunity and inflammation. We have described the pathophysiology of MPS and activated inflammation in this paper, starting with accumulating the primary storage materials, GAGs. At the initial stage of GAG accumulation, affected tissues/cells are reversibly affected but progress irreversibly to: (1) disruption of substrate degradation with pathogenic changes in lysosomal function, (2) cellular dysfunction, secondary/tertiary accumulation (toxins such as GM2 or GM3 ganglioside, etc.), and inflammatory process, and (3) progressive tissue/organ damage and cell death (e.g., skeletal dysplasia, CNS impairment, etc.). For current and future treatment, several potential treatments for MPS that can penetrate the blood-brain barrier and bone have been proposed and/or are in clinical trials, including targeting peptides and molecular Trojan horses such as monoclonal antibodies attached to enzymes via receptor-mediated transport. Gene therapy trials with AAV, ex vivo LV, and Sleeping Beauty transposon system for MPS are proposed and/or underway as innovative therapeutic options. In addition, possible immunomodulatory reagents that can suppress MPS symptoms have been summarized in this review.

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.

Oncogene-Driven Induction of Orthotopic Cholangiocarcinoma in Mice.

Cholangiocarcinoma (CCA) is a malignancy affecting the epithelial cells that line the bile ducts. This cancer shows a poor prognosis and current treatments remain inefficient. Orthotopic CCA mouse models are useful for the development of innovative therapeutic strategies. Here, we describe an orthotopic model of intrahepatic CCA that can be easily induced in mice within 5weeks at a high incidence. It is achieved by expressing two oncogenes, namely, (i) the intracellular domain of the Notch1 receptor (NICD) and (ii) AKT, in hepatocytes by means of the sleeping beauty transposon system. These plasmid vectors are delivered by hydrodynamic injection into the tail vein. The present chapter also describes how to perform magnetic resonance imaging (MRI) of the livers to visualize intrahepatic CCA nodules.

Hydrodynamic Transfection of Hepatocytes for the Study of Hepatocellular Carcinogenesis.

Hydrodynamic tail vein injection (HTVi), also called hydrodynamic gene transfer (HGT), is attracting increasing interest for modeling hepatic carcinogenesis. This highly versatile approach reproducibly provides efficient in vivo transfection of hepatocytes with naked DNA. Here, we give an in-depth description of the injection procedure, which is key for the success of the method. HTVi requires the injection of a large volume of a solution containing plasmids into the tail vein of the mouse. The transient right heart overload created by the injection forces the blood to flow back into the hepatic veins, enlarging the endothelial fenestrae and permeabilizing a fraction of hepatocytes for a few seconds. This results in the uptake of plasmids by the permeabilized hepatocytes, giving rise to their in vivo transfection. Including the Sleeping Beauty transposon system among the injected plasmids leads to the stable transfection of a subset of hepatocytes. HTVi is a powerful technique which enables numerous applications in liver cancer biology, such as a study of oncogene cooperation, of tumor heterogeneity, and interaction with the tumor microenvironment.

Optimization of a Universal Allogeneic CAR-T Cells Combining CRISPR and Transposon-Based Technologies for Treatment of Acute Myeloid Leukemia

Background: Autologous adoptive immunotherapy using T cells engineered with Chimeric Antigen Receptors (CAR-T cells) targeting CD19 and BCMA have demonstrated impressive results in B-cell malignancies, achieving long-term responses. However, similar results have not been obtained in other hematological diseases such as acute myeloid leukemia (AML). The aim of our study was to develop an optimized CAR-T product for the treatment of patients with R/R AML, that could address some of the current limitations associated with the lack of fitness of T-cells from patients with AML. Methodology: Peripheral blood samples were collected from patients diagnosed with AML and from young and aged matched healthy donors. Second generation CAR-T developed based on 2 different antibodies and modifications in the co-stimulatory and hinge domains were tested using reporter cell lines. Phenotypic and functional characterization of CAR-T generated from controls and AML T-cells were performed. CRISPR-Cas9 technology and the Sleeping Beauty transposon system were employed to generate CAR-T cells with HLA-I and TCR knockout (HLA-I KO/TCR KO). Results: We initially compared the phenotype of T cells from AML patients and healthy young and elderly controls. This analysis showed that T cells from AML patients displayed a predominantly effector phenotype, with increased expression of activation (CD69 and HLA-DR) and exhaustion markers (PD1 and LAG3), in contrast to the enriched memory phenotype observed in healthy donors. This differentiated and more exhausted phenotype was also observed, and corroborated by transcriptomic analyses, in CAR-T cells from AML patients engineered with an optimized CAR construct targeting CD33, resulting in a decreased in vivo antitumoral efficacy evaluated in xenograft AML models. To overcome some of these limitations we combined CRISPR-based genome editing technologies with virus-free gene-transfer strategies using Sleeping Beauty transposons, to generate CAR-T cells depleted of HLA-I and TCR complexes for allogeneic approaches. Our optimized protocol allows one-step generation of edited CAR-T cells that show a similar phenotypic profile than non-edited CAR-T cells, with equivalent in vitro and in vivo antitumoral efficacy. Moreover, genomic analysis of edited CAR-T cells revealed a safe integration profile of the vector, with no preferences for specific genomic regions, with a highly specific editing of the HLA-I and TCR, without significant off-target sites. Finally, production of edited CAR-T cells at a larger scale allowed the generation and selection of enough HLA-IKO/TCRKO CAR-T cells that would be compatible with clinical applications. Conclusions: In summary, our results demonstrate that CAR-T cells from AML patients, although functional, present phenotypic and functional features that could compromise their antitumoral efficacy, compared to CAR-T cells from healthy donors. The combination of CRISPR technologies with transposon-based delivery strategies allow the generation of HLA-IKO/TCRKO CAR-T cells, compatible with allogeneic approaches, that would represent a promising option for AML treatment.

CD19-CAR Cytokine Induced Killer Cells Armored with IL-18 Control Tumor Burden, Prolong Mouse Survival and Result in In Vivo Persistence of CAR-CIK Cells in a Model of B-Cell Acute Lymphoblastic Leukemia

Rationale: Cytokine induced killer cells (CIK) are a promising cancer immunotherapy. Recently CIK cells modified to express chimeric antigen receptors (CAR) target CD19 expressing hematological malignancies including B-cell acute lymphoblastic leukemia (B-ALL). Despite encouraging clinical trial results, insufficient CAR-CIK anti-tumor activity and persistence pose major obstacles towards improved efficacy in vivo. Therefore, we optimized our CAR-CIK platform by introducing two modifications to the CD19-CAR construct. The first modification “armored” CAR-CIK cells by inserting the IL-18 gene in a bicistronic DNA plasmid enabling simultaneous surface expression of the CAR protein and secretion of IL-18, which demonstrated improved CAR-T cell function. The second modification attenuated the CD28 cytoplasmic signaling domain of the CAR molecule to enhance anti-tumor activity and in vivo persistence since recent findings demonstrate that persistent or chronic CAR signaling can impair anti-tumor activity and in vivo persistence. Methods: CARCIK-1918 cells generated from donor-derived CIK cells are engineered to express CAR and IL-18 using the non-viral Sleeping Beauty (SB) transposon system. CIK cells were generated from donor PBMCs by the sequential addition of IFN-ϒ and stimulation with anti-CD3 antibody plus IL-2. After 2 days of stimulation CIK cells were co-electroporated with SB100X transposase mRNA and plasmid DNA containing a bicistronic transgene encoding both the anti-CD19 CAR and IL-18. This second-generation CAR contains a fusion of the CD28 transmembrane domain, intracellular CD28 and, TCR CD3ζ chain. In some instances, the cytoplasmic CD28 signaling domain is modified to attenuate CD28 signaling. This method of gene transfer results in stable surface expression of the CAR on CD3+ cells through a 17-day culture period. Day 17 cells are harvested, frozen and used for all subsequent studies. CARCIK-1918 cells are stimulated in vitro with CD19+ REH tumor cells and the phenotype of activated cells was characterized by multi-color flow cytometry. IL-18 secretion was determined by cytokine bead array analysis of supernatants collected post stimulation. CARCIK-1918 cells with or without the attenuated CD28 signaling domain were tested in vivo in a xenograft model of B-ALL, using NSG mice engrafted with CD19+ Raji tumor cells. Tumor burden was assessed weekly by bioluminescence imaging, the in vivo persistence of CARCIK-1918 cells was measured by flow cytometry, and survival was monitored throughout the study. Results: CARCIK-1918 cells express both IL-18 and CAR with or without attenuated CD28 signaling. Furthermore, stimulation of CARCIK-1918 cells with CD19+ REH tumor cells increases the frequency of CAR-CIK cells with a memory phenotype and concurrent upregulation of the high affinity IL-2 receptor, CD25, and the costimulatory receptor CD137. Expression of IL-18 leads to higher engraftment of CAR-CIK cells in tumor bearing NSG mice compared to unarmored controls. NSG mice engrafted with luciferase positive Raji cells demonstrated decreased bioluminescent signal following CARCIK-1918 treatment, consistent with CARCIK derived anti-leukemic activity. This anti-tumor activity corresponded to a statistically significant improvement in survival as mice treated with 2 nd generation CAR transgene CARCIK cells with or without attenuated CD28 signaling had a median survival of 37.5 (n=10; p=0.0001) and 38 (n=12; p =0.0001 days), respectively, compared to untreated mice that develop progressive highly disseminated tumor and succumb to disease by 3 weeks post tumor inoculation (n=11; median survival 18 days). Interestingly, mice treated with CARCIK-1918 cells with the CD28 attenuated signaling domain exhibited better tumor control and enhanced persistence in vivo, despite no significant improvement in survival compared to WT CD28 signaling CARCIK-1918 cells. Summary: This study demonstrates that administration of CAR-CIK cells armored with IL-18 results in a significant survival advantage for tumor challenged animals versus untreated animals. Furthermore, modifying the cytoplasmic CD28 domain to attenuate signaling, results in better control of tumor burden, and persistence of CARCIK-1918 cells in vivo. Our study is the first report of IL-18 armored CAR-CIK cells which may present a promising, novel strategy for the treatment of B-ALL.

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 & 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 >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 >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.

Understanding cancer drug resistance with Sleeping Beauty functional genomic screens: Application to MAPK inhibition in cutaneous melanoma

Combined BRAF and MEK inhibition is an effective treatment for BRAF-mutant cutaneous melanoma. However, most patients progress on this treatment due to drug resistance. Here, we applied the Sleeping Beauty transposon system to understand how melanoma evades MAPK inhibition. We found that the specific drug resistance mechanisms differed across melanomas in our genetic screens of five cutaneous melanoma cell lines. While drivers that reactivated MAPK were highly conserved, many others were cell-line specific. One such driver, VAV1, activated a de-differentiated transcriptional program like that of hyperactive RAC1, RAC1P29S. To target this mechanism, we showed that an inhibitor of SRC, saracatinib, blunts the VAV1-induced transcriptional reprogramming. Overall, we highlighted the importance of accounting for melanoma heterogeneity in treating cutaneous melanoma with MAPK inhibitors. Moreover, we demonstrated the utility of the Sleeping Beauty transposon system in understanding cancer drug resistance.

Transposon delivery for CRISPR-based loss-of-function screen in mice identifies NF2 as a cooperating gene involved with the canonical WNT signaling molecular class of hepatocellular carcinoma

Various molecular subclasses of hepatocellular carcinoma (HCC) exists, with many novel cooperating oncogenes and tumor suppressor genes involved in its tumorigenesis. The emerging importance of WNT signaling in HCC has been established. However, the intricate genetic mechanisms involved in this complex signaling pathway remains to be elucidated. Importantly, while some cooperating genes have been identified, there are still many unknown genes associated with catenin beta 1 (CTNNB1)-induced HCC. Mutations in both oncogenes and tumor suppressor genes are required for HCC tumorigenesis. The emergence of the CRISPR/Cas9 system has allowed researchers now to target both alleles efficiently. In this novel study, the Sleeping Beauty transposon system was used as a gene delivery system in vivo to stably integrate an expression cassette that carry pools of gRNAs and overexpress a mutant version of CTNNB1 into the hepatocyte genome. We identified 206 candidate genes that drive HCC tumorigenesis in the context of WNT signaling activation and, neurofibromin 2 (NF2) gene, a known tumor suppressor gene with clinical relevance was validated in this proof-of-principle study.

Optical measurement of gating pore currents in hypokalemic periodic paralysis model cells.

Hypokalemic periodic paralysis (HypoPP) is a rare genetic disease associated with mutations in CACNA1S or SCN4A, encoding Cav1.1 or Nav1.4, respectively. Most HypoPP-associated missense changes occur at the arginine residues within the voltage-sensing domain (VSD) of these channels. It is established that such mutations destroy the hydrophobic seal separating the external water and the internal cytosolic crevices, resulting in the generation of aberrant leak currents called gating pore currents. Presently, the gating pore currents are thought to underlie HypoPP. Here, we generated HEK293T-based HypoPP-model cell lines with the Sleeping Beauty transposon system that co-express mouse inward-rectifier potassium channel (mKir2.1) and HypoPP2-associated Nav1.4 variants. Whole cell patch-clamp measurements confirmed that mKir2.1 successfully hyperpolarized the membrane potential to comparable levels to myofibers, and that some Nav1.4 variants induced notable proton-based gating pore currents. Importantly, we succeeded in fluorometrically measuring the gating pore currents in these variants using a ratiometric pH indicator, SNARF-4F. Our optical method provides a potential in vitro platform for high-throughput drug screen, not only for HypoPP but also for other channelopathies caused by VSD mutations.

Enhanced Biosafety of the Sleeping Beauty Transposon System by Using mRNA as Source of Transposase to Efficiently and Stably Transfect Retinal Pigment Epithelial Cells

Neovascular age-related macular degeneration (nvAMD) is characterized by choroidal neovascularization (CNV), which leads to retinal pigment epithelial (RPE) cell and photoreceptor degeneration and blindness if untreated. Since blood vessel growth is mediated by endothelial cell growth factors, including vascular endothelial growth factor (VEGF), treatment consists of repeated, often monthly, intravitreal injections of anti-angiogenic biopharmaceuticals. Frequent injections are costly and present logistic difficulties; therefore, our laboratories are developing a cell-based gene therapy based on autologous RPE cells transfected ex vivo with the pigment epithelium derived factor (PEDF), which is the most potent natural antagonist of VEGF. Gene delivery and long-term expression of the transgene are enabled by the use of the non-viral Sleeping Beauty (SB100X) transposon system that is introduced into the cells by electroporation. The transposase may have a cytotoxic effect and a low risk of remobilization of the transposon if supplied in the form of DNA. Here, we investigated the use of the SB100X transposase delivered as mRNA and showed that ARPE-19 cells as well as primary human RPE cells were successfully transfected with the Venus or the PEDF gene, followed by stable transgene expression. In human RPE cells, secretion of recombinant PEDF could be detected in cell culture up to one year. Non-viral ex vivo transfection using SB100X-mRNA in combination with electroporation increases the biosafety of our gene therapeutic approach to treat nvAMD while ensuring high transfection efficiency and long-term transgene expression in RPE cells.

Abstract 1116: Identifying neuroendocrine prostate cancer drivers using in vivo forward genetics

Abstract Prostate cancer (PC) is the malignancy with the highest incidence worldwide in males. Although localized disease can be effectively treated and usually has a favorable prognosis, progression to metastasis results in high mortality rates as current treatment modalities are not yet curative. The standard of care for advanced PC is hormonal therapy, termed Androgen Deprivation Therapy (ADT). Unfortunately, ADT typically elicits the emergence of a lethal phenotype, termed Castration-Resistant Prostate Cancer (CRPC), which is treatment-resistant and frequently gives rise to a highly aggressive variant with features of neuroendocrine (NE) differentiation, known as Neuroendocrine PC (NEPC). There is no cure for NEPC, thus identifying drivers that lead to its emergence is of paramount importance. We used the Sleeping Beauty (SB) transposon system to perform a forward genetic screening on Genetically-Engineered Mouse Models (GEMMs) of CRPC that are known to develop NEPC under ADT pressure. SB transposon activation generated tumors with an accelerated growth phenotype and increased metastasis incidence compared to non-activated controls. Notably, several tumors presented histological features of NEPC. We sequenced SB transposon integration sites on tumors’ genomes to identify recurrent patterns suggesting genes responsible for phenotype acceleration and NEPC differentiation. Recently, we generated and assembled a set of >100 GEMM-derived tumors, that recapitulate a broad spectrum of PC disease states, to reverse-engineer a network of regulatory proteins. We used this network on RNA-Seq data collected from SB-activated tumors to identify Master Regulator (MR) proteins that are specifically associated with NEPC emergence. Cross-species network analysis of patient-specific MRs activity conservation between patient and our mouse cohorts, aligned NEPC patients to mice tumors with NE features, corroborating our study's clinical relevance. We performed an integrative network analysis that links genomic events as generated by SB transposon integrations to downstream regulatory programs implemented by MR proteins. This analysis prioritized Sirt1 as upstream modulator of NEPC MRs activity. Activation of SIRT1 in human adenocarcinoma PC cells induced NE-like phenotype, while SIRT1 inhibitor in NEPC cells reduced tumor aggressiveness. In summary, we used in vivo models of prostate cancer to elucidate molecular drivers of NEPC, a lethal variant that is treatment-resistant. Integrative network data analysis prioritized Sirt1 as driver of NEPC emergence and was validated in vivo. Citation Format: Alessandro Vasciaveo, Francisca Nunes De Almeida, Min Zou, Matteo Di Bernardo, Andrea Califano, Cory Abate-Shen. Identifying neuroendocrine prostate cancer drivers using in vivo forward genetics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1116.

310 Mechanisms of progression of myelodysplastic syndrome to fatal secondary acute myeloid leukemia

OBJECTIVES/GOALS: Aim 1: Define the genetic events that cooperate with Trp53 mutations to mediate the transformation of MDS to sAML at the stem cell level. Aim 2: Use Bayesian networks to model the signaling pathway activation states identify aberrant regulators of self-renewal in LSCs. METHODS/STUDY POPULATION: To model the transformation of MDS, I will utilize a mouse model of MDS and sAML. The Trp53 mutated mouse mimics the genetics of human MDS and develops MDS. To discover how additional mutations contribute to disease progression, I use the Sleeping Beauty transposon system which induces random mutations. I propose to use this mouse model to define the molecular mechanisms of transformation of MDS to sAML. I will also measure levels of activated signaling intermediates in sAML using CyTOF. CyTOF is a method of flow cytometry that measures up to 40 proteins simultaneously at single-cell resolution. I will also use Bayesian network to model the signaling architecture of the MDS/sAML stem cells. RESULTS/ANTICIPATED RESULTS: I will identify putative mediators of self-renewal in gene expression data and the corresponding Bayesian networks model. I will prioritize genes and signaling intermediates that act in pathways that rank highly in both methods and those that have previously published roles in self-renewal. Based on prior work, I will focus on EZH2, NFï «B, and mTOR pathways in addition to candidate pathways revealed by these studies. Small molecule inhibitors that target candidate signaling intermediates will be used for experimental validation. I will test the impact of these small molecule inhibitors on LSC self-renewal using serial colony forming assays as well as in vivo mouse reconstitution assays. DISCUSSION/SIGNIFICANCE: We focus on the role of stem cells in the transformation of MDS to sAML. Understanding the mutations and signaling relationships that mediate self-renewal in Trp53 mutant stem cells will allow for precise therapeutic target of this disease and improve outcomes for these patients.

MBTPS2 acts as a regulator of lipogenesis and cholesterol synthesis through SREBP signalling in prostate cancer

BackgroundProstate cancer is the most common cancer in men in the developed world, with most deaths caused by advanced and metastatic disease which has no curative options. Here, we identified Mbtps2 alteration to be associated with metastatic disease in an unbiased in vivo screen and demonstrated its regulation of fatty acid and cholesterol metabolism.MethodsThe Sleeping Beauty transposon system was used to randomly alter gene expression in the PtenNull murine prostate. MBTPS2 was knocked down by siRNA in LNCaP, DU145 and PC3 cell lines, which were then phenotypically investigated. RNA-Seq was performed on LNCaP cells lacking MBTPS2, and pathways validated by qPCR. Cholesterol metabolism was investigated by Filipin III staining.ResultsMbtps2 was identified in our transposon-mediated in vivo screen to be associated with metastatic prostate cancer. Silencing of MBTPS2 expression in LNCaP, DU145 and PC3 human prostate cancer cells reduced proliferation and colony forming growth in vitro. Knockdown of MBTPS2 expression in LNCaP cells impaired cholesterol synthesis and uptake along with reduced expression of key regulators of fatty acid synthesis, namely FASN and ACACA.ConclusionMBTPS2 is implicated in progressive prostate cancer and may mechanistically involve its effects on fatty acid and cholesterol metabolism.

Comprehensive Overview of CAR-T Cell Therapy, Engineering Process and Future Prospects

Chimeric antigen receptor (CAR)-T cell therapy is a revolutionary treatment method which applies the technology of modifying patients’ immune T cells to eliminate cancer cells. The immune system recognizes invading cells by noticing antigens on the foreign cells. The receptors of T cells bind to the antigens which notifies the rest of the immune system to eradicate the foreign invaders. CAR-T cell therapy has gained achievement in the treatment of hematologic malignancies such as B-ALL. CAR-T cell engineering process contains four steps including leukapheresis and the expression of the CAR on the T cells. Among the process, the Sleeping Beauty transposon system shortens the time between genetic modification and infusion so that patients can receive the modified T cells on site. GMP (Good Manufacture Practice) also ensures quality and safety of the CAR-T cells before infusing into the patients. CAR-T cells damage tumor cells by three major pathways. T cells utilize perforin and granzyme to lyse open antigen-positive tumor cells and use Fas and Fas ligand to target antigen-negative tumor cells. The derivation of cytokines from CAR-T cells sensitizes the tumor stroma and enhances tumor killing ability. The development in CAR-T cell designs has made a huge contribution to the success of the treatment where five generations of CAR-T cells have already been investigated. However, there are still some challenges associated with the treatment such as antigen escape relapse and on-target off-tumor toxicities observed in solid tumors. The technology can be further innovated by overcoming antigen escape loss, enhancing safety of CAR-T cells, and improving the persistence of CAR-T cells using the combination of oncolytic viruses with CAR-T cells. This review mainly focuses on the CAR-T cell engineering process and killing mechanisms as well as some obstacles and potential improvement for the technology.

Myb Repression Mediates Stat5b-knockdown-induced Apoptosis and Inhibits Proliferation of Glioblastoma Stem Cells.

Glioblastoma is the most common and aggressive malignant brain tumor in adults, and glioblastoma stem cells (GSCs) contribute to treatment resistance and recurrence. Inhibition of Stat5b in GSCs suppresses cell proliferation and induces apoptosis. Herein, we investigated the mechanisms of growth inhibition by Stat5b knockdown (KD) in GSCs. GSCs were established from a murine glioblastoma model in which shRNA-p53 and EGFR/Ras mutants were induced in vivo using a Sleeping Beauty transposon system. Microarray analyses were performed on Stat5b-KD GSCs to identify genes that are differentially expressed downstream of Stat5b. RT-qPCR and western blot analyses were used to determine Myb levels in GSCs. Myb-overexpressing GSCs were induced by electroporation. Proliferation and apoptosis were evaluated by a trypan blue dye exclusion test and annexin-V staining, respectively. MYB, which is involved in the Wnt pathway, was identified as a novel gene whose expression was down-regulated by Stat5b-KD in GSCs. Both MYB mRNA and protein levels were down-regulated by Stat5b-KD. Overexpression of Myb rescued cell proliferation that was suppressed by Stat5b-KD. Furthermore, Stat5b-KD-induced apoptosis in GSCs was significantly inhibited by Myb overexpression. Down-regulation of Myb mediates Stat5b-KD-induced inhibition of proliferation and induction of apoptosis in GSCs. This may represent a promising novel therapeutic strategy against glioblastoma.