Transposon Vector Research Articles

Intrahepatic Exhausted Antiviral Immunity in an Immunocompetent Mouse Model of Chronic Hepatitis B

Targeting exhausted immune systems would be a promising therapeutic strategy to achieve a functional cure for HBV infection in patients with chronic hepatitis B (CHB). However, animal models recapitulating the immunokinetics of CHB are very limited. We aimed to develop an immunocompetent mouse model of CHB for intrahepatic immune profiling. CHB mice were created by intrahepatic delivery of the Sleeping Beauty transposon vector tandemly expressing the hepatitis B virus (HBV) genome and fumarylacetoacetate hydrolase (FAH) cDNA into C57BL/6J congenic FAH knockout mice via hydrodynamic tail vein injection. We profiled the viral and intrahepatic immune kinetics in CHB mice with or without treatment with recombinant IFNα or the hepatotropic Toll-like receptor 7 agonist SA-5 using single-cell RNA-seq. CHB mice exhibited sustained HBV viremia and persistent hepatitis. They showed intrahepatic expansion of exhausted CD8+ T (Tex) cells, the frequency of which was positively associated with viral load. Recruited macrophages increased in number but impaired inflammatory responses in the liver. The cytotoxicity of mature natural killer (NK) cells also increased in CHB mice. IFNα and SA-5 treatment both resulted in viral suppression with mild hepatic flares in CHB mice. Although both treatments activated NK cells, SA-5 had the capacity to revitalize the impaired function of Tex cells and liver-recruited macrophages. Our novel CHB mouse model recapitulated the intrahepatic exhausted antiviral immunity in patients with CHB, which might be able to be reinvigorated by a hepatotropic TLR7 agonist.

Good manufacturing practice-grade generation of CD19 and CD123-specific CAR-T cells using piggyBac transposon and allogeneic feeder cells in patients diagnosed with B-cell non-Hodgkin lymphoma and acute myeloid leukemia.

The non-viral production of CAR-T cells through electroporation of transposon DNA plasmids is an alternative approach to lentiviral/retroviral methods. This method is particularly suitable for early-phase clinical trials involving novel types of CAR-T cells. The primary disadvantage of non-viral methods is the lower production efficiency compared to viral-based methods, which becomes a limiting factor for CAR-T production, especially in chemotherapy-pretreated lymphopenic patients. We describe a good manufacturing practice (GMP)-compliant protocol for producing CD19 and CD123-specific CAR-T cells based on the electroporation of transposon vectors. The lymphocytes were purified from the blood of patients undergoing chemotherapy for B-NHL or AML and were electroporated with piggyBac transposon encoding CAR19 or CAR123, respectively. Electroporated cells were then polyclonally activated by anti-CD3/CD28 antibodies and a combination of cytokines (IL-4, IL-7, IL-21). The expansion was carried out in the presence of irradiated allogeneic blood-derived mononuclear cells (i.e., the feeder) for up to 21 days. Expansion in the presence of the feeder enhanced CAR-T production yield (4.5-fold in CAR19 and 9.3-fold in CAR123). Detailed flow-cytometric analysis revealed the persistence of early-memory CAR-T cells and a low vector-copy number after production in the presence of the feeder, with no negative impact on the cytotoxicity of feeder-produced CAR19 and CAR123 T cells. Furthermore, large-scale manufacturing of CAR19 carried out under GMP conditions using PBMCs obtained from B-NHL patients (starting number=200x10e6 cells) enabled the production of >50x10e6 CAR19 in 7 out of 8 cases in the presence of the feeder while only in 2 out of 8 cases without the feeder. The described approach enables GMP-compatible production of sufficient numbers of CAR19 and CAR123 T cells for clinical application and provides the basis for non-viral manufacturing of novel experimental CAR-T cells that can be tested in early-phase clinical trials. This manufacturing approach can complement and advance novel experimental immunotherapeutic strategies against human hematologic malignancies.

Hypoxia-responsive synthetic gene circuits to improve safety and potency of CAR T cell therapy for solid tumors.

37 Background: Engineered cell therapies expressing a chimeric antigen receptor (CAR) in human T cells have demonstrated great promise for the treatment of hematological malignancies. However, unique challenges remain in translating the success of CAR T cell therapies to solid tumors, namely: (1) specificity: there are few truly unique antigens present on solid tumors, leading to off-target effects that damage normal tissue and pose safety risks; and (2) suppression: solid tumors develop microenvironments that are immunosuppressive and decrease T cell survival and tumor-directed cytotoxicity. To address these challenges and improve the safety and potency of solid tumor cell therapy, we engineered cells with synthetic genetic circuits containing a previously validated hypoxia biosensor (HBS) that responds to upregulation of the native HIF1α and HIF2α transcription factors by hypoxia, enabling the cells to sense and respond to the hypoxic tumor microenvironment ubiquitous among solid tumors. Methods: We evaluated CAR expression from circuit architectures containing the HBS promoter driving expression of both CAR and native HIF1α, HIF2α, as well as from circuit architectures containing the HBS promoter and our toolkit of synthetic transcription factors and promoters. Circuits were integrated into HEK293 cells via PiggyBac transposon vector to generate stable cell lines. Cells were subsequently cultured in normoxia (21% O2) and hypoxia (1% O2), and harvested for flow cytometric analysis to evaluate CAR surface expression across a three day time frame. Results: We identified novel circuits providing fast reporter output with minimal background signal under hypoxic conditions, and elucidated design rules that enable or restrict amplification of transgene in native HIF1α and HIF2α-containing circuits. We observed differences in CAR transgene expression in hypoxia between circuits containing HIF1α and HIF2α-mediated feedback, suggesting mechanistic differences in regulation of HIF1α and HIF2α when overexpressed. Additionally, we observed greatly amplified transgene expression in circuit topologies containing feedback mediated by our synthetic transcription factor and promoter toolkit compared to native HIF1α and HIF2α-containing circuits and cells constitutively expressing CAR. Conclusions: We present a new therapeutic technology that senses hypoxic environments and enables high-output sense and respond behaviors with therapeutic outputs. We envision that this technology will enable development of safer, more effective cell therapies for solid tumors, as well as provide new biological and mechanistic insights that can inform the future development of hypoxia-responsive cell therapies.

Conversion of anti-tissue factor antibody sequences to chimeric antigen receptor and bi-specific T-cell engager format

BackgroundThe efficacy of antibody-targeted therapy of solid cancers is limited by the lack of consistent tumour-associated antigen expression. However, tumour-associated antigens shared with non-malignant cells may still be targeted using conditionally activated-antibodies, or by chimeric antigen receptor (CAR) T cells or CAR NK cells activated either by the tumour microenvironment or following 'unlocking' via multiple antigen-recognition. In this study, we have focused on tissue factor (TF; CD142), a type I membrane protein present on a range of solid tumours as a basis for future development of conditionally-activated BiTE or CAR T cells. TF is frequently upregulated on multiple solid tumours providing a selective advantage for growth, immune evasion and metastasis, as well as contributing to the pathology of thrombosis via the extrinsic coagulation pathway.MethodsTwo well-characterised anti-TF monoclonal antibodies (mAb) were cloned into expression or transposon vectors to produce single chain (scFv) BiTE for assessment as CAR and CD28-CD3-based CAR or CD3-based BiTE. The affinities of both scFv formats for TF were determined by surface plasmon resonance. Jurkat cell line-based assays were used to confirm the activity of the BiTE or CAR constructs.ResultsThe anti-TF mAb hATR-5 and TF8-5G9 mAb were shown to maintain their nanomolar affinities following conversion into a single chain (scFv) format and could be utilised as CD28-CD3-based CAR or CD3-based BiTE format.ConclusionBecause of the broad expression of TF on a range of solid cancers, anti-TF antibody formats provide a useful addition for the development of conditionally activated biologics for antibody and cellular-based therapy.

Abstract CT170: Trial in progress: A phase 1, first in human clinical trial for T-Plex, a multiplexed, enhanced T cell receptor-engineered T cell therapy (TCR-T) for solid tumors

Abstract Checkpoint immunotherapies have revolutionized solid tumor treatment yet durably benefit a minority of patients, as they rely on endogenous anti-tumor T cells. A potential solution for patients lacking functional endogenous anti-tumor T cells is engineering their T cells with exogenous T cell receptors (TCRs) to target and kill tumor cells. Initial clinical trials with TCR engineered T cell therapies (TCR-Ts) targeted single tumor antigens on single HLA and produced partial, short-lasting responses. Solid tumors are notoriously heterogenous with highly variable antigen expression. Recent discoveries also identified HLA loss of heterozygosity in up to 40% of solid tumors, allowing tumor cells to evade T cell attack. To overcome this heterogeneity, TScan has developed T-Plex, a multiplexed cell therapy comprising 2-3 different TCR-Ts, chosen from a collection of TCR-Ts called the ImmunoBank, to target different tumor antigens on different HLA types with confirmed tumor expression. To deepen clinical responses, TCR-T cells are engineered to express CD8α/β co-receptors that, in preclinical experiments, enable CD4+ helper T cells to have >100-fold improved cytotoxicity and cytokine secretion over CD4+ cells expressing the TCR alone. Finally, to allow T cell persistence despite immunosuppressive TGF-β in the solid tumor microenvironment, TCR-T cells also express the dominant negative TGF-βreceptor, enabling ~10-fold improved proliferation in the presence of TGF- β compared to T cells expressing the TCR and CD8α/β co-receptors alone. A proprietary transposon vector with larger cargo limit enables the inclusion of these additional genes. The Phase 1 study utilizes a separate screening protocol to pre-identify patients with head and neck, cervical, anogenital cancers, NSCLC, and melanoma, any time during standard clinical care, enabling rapid enrollment into the treatment protocol upon disease progression. Screening comprises germline HLA testing, and archival tumor testing for antigen expression and exclusion of HLA loss. Treatment includes standard non-myeloablative lymphodepletion followed by one or 2 doses of T-Plex infused 28 days apart. Dose escalation under the interval 3+3 design starts with testing single TCR-Ts in dose levels 1 and 2. Thereafter, TCR-Ts are combined and escalated in dose levels 3 and 4. TCR-Ts currently in the master protocol target PRAME, MAGE-A1 or HPV16 on HLA-A*02:01 or MAGE-A1 on HLA-C*07:02. Additional TCR-Ts added to the ImmunoBank and master protocol go through dose levels 1 and 2 as single therapies before becoming available for multiplexed dose levels 3 and 4. Primary endpoints include safety, feasibility and identifying the recommended Phase 2 dose. Secondary endpoints are rates and durations of response and exploratory endpoints measure T cell activation and persistence. Two additional TCR-Ts are on track to be added to the ImmunoBank, which could allow 50-80% of common solid tumor patients to qualify for multiplexed TCR-T therapy. Citation Format: Justin Moser, Brian Pico, Brian Henick, Rom Leidner, Jared Weiss, James Isaacs, Jaspreet Grewal, Michael Hurwitz, Jim Murray, Marlyane Motta, Yun Wang, Shrikanta Chattopadhyay, Debora Barton, Gavin MacBeath, Sajeve Thomas. Trial in progress: A phase 1, first in human clinical trial for T-Plex, a multiplexed, enhanced T cell receptor-engineered T cell therapy (TCR-T) for solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT170.

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.

The future of transposon-based CAR-T cell production in immunotherapy

Chimeric Antigen Receptor (CAR) T-cell therapy, being one of the most critical treatment regimens for certain types of lymphomas, leukemias, and multiple myeloma, is experiencing ever-increasing demand. Its high expense and low availability have inflicted many issues among patients in need of this treatment. This paper aims to address these socioeconomic problems by concentrating on the future and current development of transposon-based CAR-T cell production in immunotherapy. Switching viral vectors to transposon-based vectors will greatly decrease the cost and manufacturing time of this personalized product. Transposon vectors also have advantages over viral vectors in sense of DNA carrying capacity and safety matters. Research organizations such as the CARAMBA Project are undertaking new transposon technology in the hope to increase the accessibility of CAR-T cell immunotherapies. Transposon-based CAR-T cell production may pave way for therapeutically and financially satisfying immunotherapies.

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.

Production of retroviral vectors in continuous high cell density culture

Retroviral vectors derived from murine leukemia virus (MLV) are used in somatic gene therapy applications e.g. for genetic modification of hematopoietic stem cells. Recently, we reported on the establishment of a suspension viral packaging cell line (VPC) for the production of MLV vectors. Human embryonic kidney 293-F (HEK293-F) cells were genetically modified for this purpose using transposon vector technology. Here, we demonstrate the establishment of a continuous high cell density (HCD) process using this cell line. First, we compared different media regarding the maximum achievable viable cell concentration (VCC) in small scale. Next, we transferred this process to a stirred tank bioreactor before we applied intensification strategies. Specifically, we established a perfusion process using an alternating tangential flow filtration system. Here, VCCs up to 27.4E + 06 cells/mL and MLV vector titers up to 8.6E + 06 transducing units/mL were achieved. Finally, we established a continuous HCD process using a tubular membrane for cell retention and continuous viral vector harvesting. Here, the space-time yield was 18-fold higher compared to the respective batch cultivations. Overall, our results clearly demonstrate the feasibility of HCD cultivations for high yield production of viral vectors, especially when combined with continuous viral vector harvesting.Key points• A continuous high cell density process for MLV vector production was established• The tubular cell retention membrane allowed for continuous vector harvesting• The established process had a 18-fold higher space time yield compared to a batch

Anti-CD117 CAR T cells incorporating a safety switch eradicate human acute myeloid leukemia and hematopoietic stem cells

Discrimination between hematopoietic stem cells and leukemic stem cells remains a major challenge for acute myeloid leukemia immunotherapy. CAR Tcells specific for the CD117 antigen can deplete malignant and healthy hematopoietic stem cells before consolidation with allogeneic hematopoietic stem cell transplantation in absence of cytotoxic conditioning. Here we exploit non-viral technology to achieve early termination of CAR Tcell activity to prevent incoming graft rejection. Transient expression of an anti-CD117 CAR by mRNA conferred Tcells the ability to eliminate CD117+ targets invitro and invivo. As an alternative approach, we used a Sleeping Beauty transposon vector for the generation of CAR Tcells incorporating an inducible Caspase 9 safety switch. Stable CAR expression was associated with high proportion of T memory stem cells, low levels of exhaustion markers, and potent cellular cytotoxicity. Anti-CD117 CAR Tcells mediated depletion of leukemic cells and healthy hematopoietic stem cells in NSG mice reconstituted with human leukemia or CD34+ cord blood cells, respectively, and could be terminated invivo. The use of a non-viral technology to control CAR Tcell pharmacokinetic properties is attractive for a first-in-human study in patients with acute myeloid leukemia prior to hematopoietic stem cell transplantation.

PBLAM1-x: standardized transposon tools forhigh-throughput screening.

The engineering of pre-defined functions in living cells requires increasingly accurate tools as synthetic biology efforts become more ambitious. Moreover, the characterization of the phenotypic performance of genetic constructs demands meticulous measurements and extensive data acquisition for the sake of feeding mathematical models and matching predictions along the design-build-test lifecycle. Here, we developed a genetic tool that eases high-throughput transposon insertion sequencing (TnSeq): the pBLAM1-x plasmid vectors carrying the Himar1 Mariner transposase system. These plasmids were derived from the mini-Tn5 transposon vector pBAMD1-2 and built following modular criteria of the Standard European Vector Architecture (SEVA) format. To showcase their function, we analyzed sequencing results of 60 clones of the soil bacterium Pseudomonas putida KT2440. The new pBLAM1-x tool has already been included in the latest SEVA database release, and here we describe its performance using laboratory automation workflows. Graphical Abstract.

A mammalian cell display platform based on scFab transposition.

In vitro display technologies have been successfully utilized for the discovery and evolution of monoclonal antibodies (mAbs) for diagnostic and therapeutic applications, with phage display and yeast display being the most commonly used platforms due to their simplicity and high efficiency. As their prokaryotic or lower eukaryotic host organisms typically have no or different post-translational modifications, several mammalian cell-based display and screening technologies for isolation and optimization of mAbs have emerged and are being developed. We report here a novel and useful mammalian cell display platform based on the PiggyBac transposon system to display mAbs in a single-chain Fab (scFab) format on the surface of HEK293F cells. Immune rabbit antibody libraries encompassing ~7×107 independent clones were generated in an all-in-one transposon vector, stably delivered into HEK293F cells and displayed as an scFab with rabbit variable and human constant domains. After one round of magnetic activated cell sorting and two rounds of fluorescence activated cell sorting, mAbs with high affinity in the subnanomolar range and cross-reactivity to the corresponding human and mouse antigens were identified, demonstrating the power of this platform for antibody discovery. We developed a highly efficient mammalian cell display platform based on the PiggyBac transposon system for antibody discovery, which could be further utilized for humanization as well as affinity and specificity maturation.

In Vivo Sampling of Intracellular Heterogeneity of Pseudomonas putida Enables Multiobjective Optimization of Genetic Devices.

The inner physicochemical heterogeneity of bacterial cells generates three-dimensional (3D)-dependent variations of resources for effective expression of given chromosomally located genes. This fact has been exploited for adjusting the most favorable parameters for implanting a complex device for optogenetic control of biofilm formation in the soil bacterium Pseudomonas putida. To this end, a DNA segment encoding a superactive variant of the Caulobacter crescendus diguanylate cyclase PleD expressed under the control of the cyanobacterial light-responsive CcaSR system was placed in a mini-Tn5 transposon vector and randomly inserted through the chromosome of wild-type and biofilm-deficient variants of P. putida lacking the wsp gene cluster. This operation delivered a collection of clones covering a whole range of biofilm-building capacities and dynamic ranges in response to green light. Since the phenotypic output of the device depends on a large number of parameters (multiple promoters, RNA stability, translational efficacy, metabolic precursors, protein folding, etc.), we argue that random chromosomal insertions enable sampling the intracellular milieu for an optimal set of resources that deliver a preset phenotypic specification. Results thus support the notion that the context dependency can be exploited as a tool for multiobjective optimization, rather than a foe to be suppressed in Synthetic Biology constructs.

Prolonged activity of the transposase helper may raise safety concerns during DNA transposon-based gene therapy

DNA transposon-based gene delivery vectors represent a promising new branch of randomly integrating vector development for gene therapy. For the side-by-side evaluation of the piggyBac and Sleeping Beauty systems-the only DNA transposons currently employed in clinical trials-during therapeutic intervention, we treated the mouse model of tyrosinemia type I with liver-targeted gene delivery using both transposon vectors. For genome-wide mapping of transposon insertion sites we developed a new next-generation sequencing procedure called streptavidin-based enrichment sequencing, which allowed us to identify approximately one million integration sites for both systems. We revealed that a high proportion of piggyBac integrations are clustered in hot regions and found that they are frequently recurring at the same genomic positions among treated animals, indicating that the genome-wide distribution of Sleeping Beauty-generated integrations is closer to random. We also revealed that the piggyBac transposase protein exhibits prolonged activity, which predicts the risk of oncogenesis by generating chromosomal double-strand breaks. Safety concerns associated with prolonged transpositional activity draw attention to the importance of squeezing the active state of the transposase enzymes into a narrower time window.

Transgenic mammalian salivary cells expressing ligninase as a proof-of-concept model for enhanced lignocellulose degradation to generate future resilient livestock

The major limitation in feed grade utility of cereal straws is the presence of the high amount of lignified fiber. Considering the limited ligninase availability in rumen, the current work is intended to establish the proof-of-concept for a recombinant ligninase expression in mammalian salivary gland cells so that it can be translated into generation of a live transgenic ruminant that can produce ligninase in its saliva for enhanced degradation of lignin. To verify the proof of concept the Sleeping Beauty transposon vector (pT2-RMCE) containing a ubiquitously active CAGGS promoter was used to express three different types of bacterial ligninases. Two of them are dye decolorizing peroxidase family (DyP1 and Dyp2) which belong to the category of heme peroxidases and one is laccase which belongs to a multi-copper oxidase (cotA). The vector was transfected into rat ParC10 salivary cell lines and the protein expressed by the engineered cells was harvested, purified to check the functionality on Kraft lignin degradation. Among the three, only DyP1 could be detected on Western blot and was also found to be functionally active. It can be concluded that mammalian salivary cells can be genetically engineered with appropriate ligninase gene to produce functional ligninase.

Construction of a Set of Novel Transposon Vectors for Efficient Silencing of Protein and lncRNA Genes via CRISPR Interference

In recent years, CRISPR interference (CRISPRi) technology of gene silencing has emerged as a promising alternative to RNA interference (RNAi) surpassing the latter in terms of efficiency and accuracy. Here, we describe the construction of a set of transposon vectors suitable for constitutive or tetracycline (doxycycline)-inducible silencing of genes of interest via CRISPRi method and conferring three different antibiotic resistances, using vectors available via Addgene repository. We have analyzed the performance of the new vectors in the silencing of mouse Adam10 and human lncRNA, NORAD. The empty vector variants can be used to efficiently silence any genes of interest.

Early Subcellular Hepatocellular Alterations in Mice Post Hydrodynamic Transfection: An Explorative Study.

Hydrodynamic transfection (HT) or hydrodynamic tail vein injection (HTVi) is among the leading technique that is used to deliver plasmid genes mainly into the liver of live mice or rats. The DNA constructs are composed of coupled plasmids, while one contains the gene of interest that stably integrate into the hepatocyte genome with help of the other consisting sleeping beauty transposase system. The rapid injection of a large volume of DNA-solution through the tail vein induces an acute cardiac congestion that refluxed into the liver, mainly in acinus zone 3, also found through our EM study. Although, HT mediated hydrodynamic force can permeabilizes the fenestrated sinusoidal endothelium of liver, but the mechanism of plasmid incorporation into the hepatocytes remains unclear. Therefore, in the present study, we have hydrodynamically injected 2 mL volume of empty plasmid (transposon vector) or saline solution (control) into the tail vein of anesthetized C57BL/6J/129Sv mice. Liver tissue was resected at different time points from two animal group conditions, i.e., one time point per animal (1, 5, 10-20, 60 min or 24 and 48 hrs after HT) or multiple time points per animal (0, 1, 2, 5, 10, 20 min) and quickly fixed with buffered 4% osmium tetroxide. The tissues fed with only saline solution was also resected and fixed in the similar way. EM evaluation from the liver ultrathin sections reveals that swiftly after 1 min, the hepatocytes near to the central venule in the acinus zone 3 shows cytoplasmic membrane-bound vesicles. Such vesicles increased in both numbers and size to vacuoles and precisely often found in the proximity to the nucleus. Further, EM affirm these vacuoles are also optically empty and do not contain any electron dense material. Although, some of the other hepatocytes reveals sign of cell damage including swollen mitochondria, dilated endoplasmic reticulum, Golgi apparatus and disrupted plasma membrane, but most of the hepatocytes appeared normal. The ultrastructural findings in the mice injected with empty vector or saline injected control mice were similar. Therefore, we have interpreted the vacuole formation as nonspecific endocytosis without specific interactions at the plasma membrane.

Isolation and Functional Analysis of Myoepithelial Cells from Adult Mouse Submandibular Glands.

Salivary gland myoepithelial cells regulate salivary secretion and have been implicated in the histological diversity of salivary gland tumors. However, isolation of myoepithelial cells has been difficult owing to a lack of detailed functional analysis and cell surface markers. Therefore, we aimed to isolate myoepithelial cells from adult mouse submandibular glands using the epithelial marker EpCAM and cell adhesion factor CD49f as indicators and characterize them via sphere-forming culture. Functional analysis of specific gene expression in myoepithelial cells is possible via cell transfection experiments using the piggyBac transposon vector system. Here, we describe detailed methods and tips for the isolation and functional analysis of myoepithelial cells.

Genetic Manipulation of Mycoplasma pneumoniae.

Mycoplasma pneumoniae is a small cell wall-lacking bacterium that is a common cause of bronchitis and pneumonia in humans. In addition to its clinical importance, M. pneumoniae has recently been considered a promising model organism for synthetic biology because of its small genome size and unique cell structure. At one cell pole, M. pneumoniae forms the attachment organelle that is responsible for adherence to host cells and gliding motility. The attachment organelle is a membrane protrusion and is composed of number of molecules, including adhesin and cytoskeletal proteins. Genetic manipulation techniques are key research approaches for understanding the structure and the function of this unique molecular machinery. In this chapter, standard genetic engineering methods for this species using the Tn4001 transposon vector are described.

Targeted Genomic Insertion of Cars in iPSC-Derived Macrophages Leads to Improved Expression and Anti-Tumor Activity

Expression of chimeric antigen receptors (CARs) provides an important strategy to improve the anti-tumor activity of immune cells. T cells equipped with CARs specific to tumor antigens have been very effective for treatment of B cell malignancies. However, CAR-T cell-mediated treatment had limited success in solid tumors. Since macrophages are capable of infiltrating into tumor microenvironment, detecting phagocytic targets and engulfing them, as well as stimulating other endogenous immune cells, macrophages engineered with CAR constructs have been gaining interest for cancer immunotherapy. We have successfully generated CAR-expressing induced pluripotent stem cells (iPSCs) through either PiggyBac transposon-mediated transfection or lentiviral transduction. Subsequently, the CAR-expressing iPSCs were differentiated into macrophages (iPSC-CARMACs). The CAR expression in undifferentiated iPSCs were high and stable, but gradually downregulated during the differentiation into macrophages. Due to the loss of CAR expression, these iPSC-CARMACs only modestly improved phagocytic activity compared to wild-type iPSC-derived macrophages (iPSC-MACs) in vitro. Additionally, we did not find a significant benefit for in vivo anti-tumor activity of the iPSC-CARMACs compared to iPSC-MACs. We hypothesized the deficiency of anti-tumor activity might be due to the loss of CAR-expression that can become silenced after random integration into the genome. Therefore, to test this hypothesis as well as improve and stabilize the CAR expression, we engineered human iPSCs using CRISPR-Cas9 mediated CAR insertion into AAVS1 locus, a known safe harbor locus that is less prone to genetic silencing. We engineered a series of CAR constructs containing the same anti-Mesothelin (Meso) extracellular single-chain antibody variable fragment, combined with different intracellular signaling domains including Bai1, MegF10, MerTK and CD3ζ. To determine the phagocytic activity of the anti-Meso CARs expressing these different intracellular domains, we initially introduced CAR constructs into THP-1 cells. We tested these CAR-expressing THP-1-derived macrophages in phagocytosis assay with Meso-expressing A1847 ovarian cancer cells. We found Bai1 and MegF10 triggered higher levels of engulfment of tumor cells than other CAR constructs either by themselves or when combined with anti-CD47 antibody (Figure A). Given the potential anti-tumor activity of anti-Meso-Bai1 and anti-Meso-MegF10 CARs, we next generated anti-Meso-Bai1 and anti-Meso-MegF10 CAR-expressing iPSC-CARMACs by targeting at AAVS1 locus to improve CAR expression compared to the previous iPSC-CARMACs made by transposon transfection or lentiviral transduction. We successfully obtained iPSCs expressing anti-Meso-Bai1 CAR integrated into AAVS1 site (AAVS1-Meso-Bai1-iPSCs). We have differentiated the AAVS1-Meso-Bai1-iPSCs to macrophages (Meso-Bai1-CARMACs) and demonstrated Meso-Bai1-CARMACs significantly improved phagocytic activity compared to iPSC-MACs with or without the addition of anti-CD47 antibody (Figure B). Notably, CAR expression was stably maintained at a high level on the mature macrophages generated from the AAVS1-Meso-Bai1 iPSCs compared to those either from transposon or lentiviral vector mediated CAR expressing iPSCs (Figure C). We isolated two homozygous clones of AAVS1-Meso-Bai1 iPSCs that have even stronger CAR expression than bulk population of AAVS1-Meso-Bai1 iPSCs. The differentiation of these single clones, as well as the evaluation of their in vitro activity are in progress. We are also testing the in vivo function of these new AAVS1-iPSC-CARMACs in an ovarian tumor model. Together, these studies demonstrate that we can successfully produce CAR-expressing iPSC derived macrophages, which exhibit improved anti-tumor activity compared to wild-type iPSC derived macrophages. CAR expression can be best maintained on macrophages by AAVS1-locus targeting insertion. Importantly, these studies also demonstrate that strategies used to genetically modify iPSC-derived cells may need to be modified or optimized depending on which mature cell type of interest is being produced. That is, while randomly integrated CARs seem to work well in iPSC-derived NK cells and T cells, this targeted insertion approach is likely needed for derivation of more functional iPSC-derived CAR-macrophages. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal