Transposon System Research Articles

Recombinant CHO Cell Pool Generation Using PiggyBac Transposon System.

CHO cell pools with desirable characteristics of high titer and consistent product quality are useful for rapid production of recombinant proteins. Here, we describe the generation of CHO cell pools using the piggyBac transposon system for mediating gene integration. The method describes the co-transfection of cells with the donor plasmid (coding for the gene of interest) and the helper plasmid (coding for the transposase) using polyethyleneimine (PEI). This is followed by a genetic selection for the generation of a cell pool. The resulting cell pool can be used to start a batch or fed-batch culture. Alternatively, it can be used for generation of clonal cell lines or generation of cell banks for future use.

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.

A Versatile Method for Inducible Protein Production in 293 Cells Usingthe PiggyBac Transposon System.

The production of recombinant proteins has helpedinunderstanding of their function and developing newtherapies. However,one of the major bottlenecks for protein production is the establishment of reliable mammalian cell lines with high expression levels. In this chapter, we describe a simple and robust system that allows for the quick establishmentof stable transgenic 293 cell lines withreproducible and high protein expression levels. This methodology is based on the piggyBac transposon system and enablestheinducible production of the protein of interest. Finally,this methodology can easily be used in conventional laboratory cell culture settings without requiring specialized devices.

MRNA‐based chimeric antigen receptor T cell therapy: Basic principles, recent advances and future directions

AbstractNon‐viral vector chimeric antigen receptor (CAR)‐T cells have garnered increasing attention due to their ability to efficiently eradicate cancer cells while mitigating undesirable side effects. However, the current methods for engineering chimeric antigen receptor T (CAR‐T) cells employ viral vectors that result in permanent CAR expression and potentially severe negative impacts. As a solution to these challenges, triggering transitory expression of CARs in T cells via messenger RNA (mRNA) has emerged as a promising strategy. Currently, electroporation is a common method used to introduce the mRNA encoding the CAR into the T cells. Moreover, there has been increasing attention on the exploration of innovative mRNA delivery systems, including lipid, polymer‐based nanoparticle, exosomes and peptide transduction domains. Additionally, we also explored the functions of different types of mRNA in mRNA‐based CAR‐T cell therapy. The auxiliary mRNA, exemplified by systems such as megaTAL and nuclease transposon systems, demonstrates its capacity to extend CAR‐T cell viability and survival. This perspective offers the current state of mRNA‐based CAR‐T cell therapy and provides valuable insights into future research avenues.

Metagenomic comparison of effects of mesophilic and thermophilic manure anaerobic digestion on antimicrobial resistance genes and mobile genetic elements

This study evaluated the long-term effects of mesophilic (MAD) and thermophilic (TAD) anaerobic digestion on the fate and evolution of antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs) in cattle manure following a metagenomic approach. The results indicated that MAD and TAD lowered ARG levels in fresh cattle manure by over 50 % and MGEs by over 65 %. However, no statistically significant difference was observed between the MAD and TAD processes in terms of their ability to reduce ARG and MGE. Co-occurrence analysis suggested facultative anaerobic species belonging to the Bacillota and Actinomycetota phyla as the primary potential hosts of ARGs and MGEs. Analyses of co-assembled contigs revealed the presence of transposon and toxin-antitoxin systems in close proximity to ARGs typically found in anaerobic digesters. The identification of these systems near ARGs is particularly significant, as it highlights a potential mechanism for the persistence and spread of ARGs in such environments. Overall, the results indicated that digested cattle manure (whether under mesophilic or thermophilic conditions) would pose a much lower risk of dissemination of antimicrobial resistance than untreated manures. Since TAD did not outperform MAD at reducing ARGs and MGEs, thermophilic temperatures may not be necessary to improve ARG reduction rates in cattle manure AD. These valuable insights could help develop strategies to reduce the dissemination of antibiotic resistance due to manure treatment and disposal.

A highly efficient transposon vector system for recombinant protein expression in CHO cells

Chinese hamster ovary (CHO) cells are the most widely used mammalian host cells for the production of recombinant therapeutic proteins. Expression vectors are a key factor in determining the expression level of gene of interest (GOI) in CHO cells. Here, two transposon vector systems were constructed by adding two different pairs of inverted terminal repeats (ITR-1 and ITR-2), and four proteins of interest (POI), including enhanced green fluorescence protein (eGFP), secreted alkaline phosphatase (SEAP), human serum albumin (HSA) and Adalimumab (Adm) were expressed in CHO cells. The stably transfected cell pools and single cell clones were screened, and the expression levels of POIs were detected by qPCR, Western blot and ELISA. The results showed that both transposon vectors could improve the expression levels of POI at both mRNA and protein levels, while the ITR-2 sequence was more effective. Moreover, the positive cell clone rate of transposon system was higher than that of the control. In conclusion, the ITR-2 transposon vector system can be effectively used for the expression of recombinant proteins in CHO cells.

Parallelized engineering of mutational models using piggyBac transposon delivery of CRISPR libraries

New technologies and large-cohort studies have enabled novel variant discovery and association at unprecedented scale, yet functional characterization of these variants remains paramount to deciphering disease mechanisms. Approaches that facilitate parallelized genome editing of cells of interest or induced pluripotent stem cells (iPSCs) have become critical tools toward this goal. Here, we developed an approach that incorporates libraries of CRISPR-Cas9 guide RNAs (gRNAs) together with inducible Cas9 into a piggyBac (PB) transposon system to engineer dozens to hundreds of genomic variants in parallel against isogenic cellular backgrounds. This method empowers loss-of-function (LoF) studies through the introduction of insertions or deletions (indels) and copy-number variants (CNVs), though generating specific nucleotide changes is possible with prime editing. The ability to rapidly establish high-quality mutational models at scale will facilitate the development of isogenic cellular collections and catalyze comparative functional genomic studies investigating the roles of hundreds of genes and mutations in development and disease.

Influence of three different manure treatments on antimicrobial resistance genes and mobile genetic elements

There is a growing concern about the potential dissemination of antimicrobial resistance into agricultural fields due to the application of manure as crop fertilizer. While reducing the usage of antibiotics in livestock production stands as the first alternative to prevent this problem, there is evidence that this may not be enough to eliminate antimicrobial resistance elements already present in manure microbiomes. This study employed a metagenomic approach to investigate the impact of common manure treatments, including aerobic storage, mesophilic anaerobic digestion (MAD), and solid-liquid separation, on the presence and abundance of antimicrobial resistance genes (ARGs), bactericides, and heavy metal resistance genes (BacMet), and mobile genetic elements (MGEs) in manure from three different farms, including one operating in an antibiotic-free environment. The results indicated that MAD was the best method to reduce the numbers of ARGs, BacMet, and MGEs, achieving reduction rates greater than 40%, 89%, and 68%, respectively. Manure storage significantly reduced BacMet levels (over 30%) and MGEs (28%) but had no significant effect on total ARG levels. Solids recovered through solid-liquid separation exhibited elevated levels of ARGs, BacMet, and MGEs, while the liquid fraction displayed levels similar to untreated manures. Correlation and co-occurrence modeling analyses indicated that changes in microbial communities, particularly fluctuations in aerobic and facultative communities belonging to Bacillota, Actinomycetota, and Pseudomonadota phyla, played a significant role in driving changes in ARGs, BacMet, and MGEs. The results also showed the presence of toxin-antitoxin and transposon systems near different ARGs. Overall, the results confirmed that genes conferring resistance to various antimicrobials and MGE capable of mobilizing them are widely spread in dairy farms; that even under the absence of antibiotics, the use of heavy metals and disinfectants may promote the maintenance of ARGs and MGEs, and; that treatment such as anaerobic digestion could reduce the risk of the spread of antimicrobial resistance.

NK92 Expressing Anti-BCMA CAR and Secreted TRAIL for the Treatment of Multiple Myeloma: Preliminary In Vitro Assessment.

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

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.

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.

Efficacy of Human iPSC-Derived CAR-NK Cells Targeting Multiple Myeloma Cells

NK cells armed with chimeric antigen receptors (CAR) enable NK cells specifically to target cancer cells by recognizing tumor associated antigens. Previous studies have proven that human iPSC-derived CAR-NK cells have enhanced anti-tumor activity. Targeted B-cell maturation antigen (BCMA) therapy for relapsed refractory MM (rrMM) has been widely used with antibody-drug conjugate (ADC), bispecific antibodies, and CART cells, which has produced a rapid response, but relapse is common. Recent work has revealed GPRC5D as an alternative target in MM for immunotherapy and patients who have previously received anti-BCMA therapy have responded to GPRC5D CART cells. To assess target GPRC5D therapy with CAR-NK cells, which may in future serve as allogeneic immunotherapy, we transduced anti-GPRC5D CAR into an iPSC line derived from a healthy donor using a piggybac transposon system. More than 97% of CAR inserted-iPSCs (CAR-iPSCs) expressed anti-GPRC5D scFV with a similar expression level identified in CAR-iPSC-derived-NK cells (CAR-iNK, 92.7%). The anti-GPRC5D CAR-iNK demonstrated high purity (>99.9% for CD45 + and CD56 +) and expressed a high level of CD16 (63.6%), the NK cell activating receptor NKG2D (96.3%) and NKp30 (98.7%), and the co-stimulatory receptors CD244 (99.6%) and CD226 (97.8%). Nonetheless expression of TCR⍺β and TCRƳ- was relatively low (<2%). Cytotoxicity assay revealed that anti-GPRC5D CAR-iNK had similar cytotoxicity against K562 cells (No GPRC5D antigen) to cord blood-derived NK cells (CB-NK), wide type (WT) iNK and anti-BCMA CAR-iNK. Cytotoxicity was dose-dependent indicating that these NK cells had similar innate anti-cancer cytotoxicity. When incubated at a ratio of 4:1 with NCI-H929 cells (MM) for 4 hours, which express a high level of both BCMA and GPRC5D, anti-GPRC5D CAR-iNK demonstrated an overwhelming advantage (approximately 90% killing rate) over the CB-NK (no killing) and WT iNK (around 10% killing rate). Furthermore, the anti-GPRC5D CAR-iNK also showed superior cytotoxicity to anti-BCMA CAR-iNK (Figure 1), indicating that GPRC5D may be a more efficient target for CAR-NK-based immunotherapy in MM. We further confirmed that anti-GPRC5D CAR-NK have acceptable cytotoxicity to NCI-H929 and OPM-2 cells, even when transported over a long distance (more than 2000 km) and following cryopreservation. The lower expression by OPM-2 cells of BCMA and GPRC5D compared with NCI-H929 indicates that both fresh and cryopreserved anti-GPRC5D CAR-iNK can specifically target MM with a higher and lower expression of GPRC5D. In addition, cryopreserved anti-GPRC5D CAR-iNK cells maintain comparable antigen-specific cytotoxicity in vitro, and demonstrate the ability to reduce tumor burden in an antigen-specific manner in an OPM-2 xenograft model. Our results demonstrate that genome-engineered human iPSCs can provide an unlimited source for manufacturing scalable, off-the-shelf, and cost-effective CAR-NK cells, and GPRC5D is a potent immunotherapeutic target in multiple myeloma.

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.

PiggyBac-mediated genomic integration of linear dsDNA-based library for deep mutational scanning in mammalian cells.

Deep mutational scanning (DMS) makes it possible to perform massively parallel quantification of the relationship between genetic variants and phenotypes of interest. However, the difficulties in introducing large variant libraries into mammalian cells greatly hinder DMS under physiological states. Here, we developed two novel strategies for DMS library construction in mammalian cells, namely 'piggyBac-in vitro ligation' and 'piggyBac-in vitro ligation-PCR'. For the first strategy, we took the 'in vitro ligation' approach to prepare high-diversity linear dsDNAs, and integrate them into the mammalian genome with a piggyBac transposon system. For the second strategy, we further added a PCR step using the in vitro ligation dsDNAs as templates, for the construction of high-content genome-integrated libraries via large-scale transfection. Both strategies could successfully establish genome-integrated EGFP-chromophore-randomized libraries in HEK293T cells and enrich the green fluorescence-chromophore amino-acid sequences. And we further identified a novel transcriptional activator peptide with the 'piggyBac-in vitro ligation-PCR' strategy. Our novel strategies greatly facilitate the construction of large variant DMS library in mammalian cells, and may have great application potential in the future.

Mosquito Embryo Microinjection.

Genetically modified (GM) mosquitoes are an important tool in the fight against mosquito-borne disease, both indirectly through their use in research investigating host-pathogen interaction, mosquito olfaction, and anthropomorphic behavior and in future direct uses for suppression and possibly eradication through sterile insect technique (SIT) and/or gene-drive programs. Successful creation of GM mosquitoes depends on microinjection procedures that precisely deliver injection materials while causing as little damage to mosquito embryos as possible. Genetic modification reagents, such as transposon system components (vector plasmids, helper plasmids, and helper mRNA), and CRISPR-Cas9 components (guide RNAs, Cas9 protein, plasmids expressing Cas9 and/or guide RNAs, and donor plasmids used in homology-directed repair [HDR]), must be delivered into the preblastoderm embryo at the posterior end where the pole cells will form before cellularization occurs. Sharp needles that pierce the embryo easily are important tools in this procedure and work best when the embryos are not desiccated. The two main procedures for mosquito embryo microinjection involve injecting embryos under halocarbon oil or under aqueous solution.

The neuroprotective effects of FG-4592, a hypoxia-inducible factor-prolyl hydroxylase inhibitor, against oxidative stress induced by alpha-synuclein in N2a cells

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra. The pathological hallmark of PD is the appearance of intraneuronal cytoplasmic α-synuclein (α-Syn) aggregation, called Lewy bodies. α-Syn aggregation is deeply involved in the pathogenesis of PD. Oxidative stress is also associated with the progression of PD. In the present study, to investigate whether a hypoxia-inducible factor (HIF)-prolyl hydroxylase (PH) inhibitor, FG-4592 (also called roxadustat), has neuroprotective effects against α-Syn-induced neurotoxicity, we employed a novel α-Syn stably expressing cell line (named α-Syn-N2a cells) utilizing a piggyBac transposon system. In α-Syn-N2a cells, oxidative stress and cell death were induced by α-Syn, and FG-4592 showed significant protection against this neurotoxicity. However, FG-4592 did not affect α-Syn protein levels. FG-4592 triggered heme oxygenase-1 (HO-1) expression downstream of HIF-1α in a concentration-dependent manner. In addition, FG-4592 decreased the production of reactive oxygen species possibly via the activation of HO-1 and subsequently suppressed α-Syn-induced neurotoxicity. Moreover, FG-4592 regulated mitochondrial biogenesis and respiration via the induction of the peroxisome proliferator-activated receptor-γ coactivator-1α. As FG-4592 has various neuroprotective effects against α-Syn and is involved in drug repositioning, it may have novel therapeutic potential for PD.

Ligand-based, piggyBac-engineered CAR-T cells targeting EGFR are safe and effective against non-small cell lung cancers

Epidermal growth factor receptor (EGFR) is overexpressed in various cancers, including non-small cell lung cancer (NSCLC), and in some somatic cells at a limited level, rendering it an attractive antitumor target. In this study, we engineered chimeric antigen receptor (CAR)-T cells using the piggyBac transposon system, autologous artificial antigen-presenting cells, and natural ligands of EGFR. We showed that this approach yielded CAR-T cells with favorable phenotypes and CAR positivity. They exhibited potent antitumor activity against NSCLC both invitro and invivo. When administered to tumor-bearing mice and non-tumor-bearing cynomolgus macaques, they did not elicit toxicity despite their cross-reactivity to both murine and simian EGFRs. In total we tested three ligands and found that the CAR candidate with the highest affinity consistently displayed greater potency without adverse events. Taken together, our results demonstrate the feasibility and safety of targeting EGFR-expressing NSCLCs using ligand-based, piggyBac-engineered CAR-T cells. Our data also show that lowering the affinity of CAR molecules is not always beneficial.

Transcriptomic Analysis of Insulin-Secreting Murine Hepatocytes Transduced with an Integrating Adeno-Associated Viral Vector

Type 1 Diabetes (T1D) is a chronic metabolic disorder for which current treatments are unable to prevent the onset of complications. Previously, we used an adeno-associated viral vector (AAV8) to deliver furin-cleavable human insulin (INS-FUR) to the livers of diabetic non-obese diabetic (NOD) mice to reverse T1D. The use of the traditional AAV8-INS-FUR vector could not bring about normoglycemia. However, this vector, coupled with a transposon system in the AAV8/piggyBac-INS-FUR vector, was able to do so. This study aimed to investigate the transcriptomic profiles of the livers of diabetic, AAV8-INS-FUR-transduced, and AAV8/piggyBac-INS-FUR-transduced NOD mice and compare these to the normal liver to identify genetic differences resulting from delivery of the AAV8/piggyBac-INS-FUR vector which produced normoglycemia. Differential gene expression was determined by RNA-Seq analysis and differentially expressed genes from each treatment were mapped onto cellular pathways to determine the treatments’ cell signaling and downstream effects. We observed distinct differences between the piggyBac-transduced and diabetic models, particularly in terms of metabolic function and the upregulation of key pancreatic markers in the liver of piggyBac-transduced animals. The success of the AAV8/piggyBac-INS-FUR vector in achieving normoglycemia through stable transduction was evident. However, further engineering is necessary to achieve complete pancreatic transdifferentiation of liver cells.

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.