piggyBac Research Articles

EGFRvIII-specific CAR-T cells produced by piggyBac transposon exhibit efficient growth suppression against hepatocellular carcinoma.

Adoptive cellular immunotherapy employing chimeric antigen receptors-modified T (CAR-T) cells has demonstrated promising antitumor effects in hematologic cancers. However, CAR-T therapy confront many challenges in solid tumors like immunosuppressive microenvironment, molecular heterogeneity, etc. The cancer genome atlas (TCGA) of hepatocellular carcinoma (HCC) revealed many genetic characteristic and molecular tumorigenesis. EGFRvIII is a tumor specific antigen widely expressed in a variety of cancers including HCC and an ideal therapeutic target for cancer therapy. The liver cancer cell line SMMC7721 express high level EGFRvIII and widely applied in HCC investigations. Herein, we developed EGFRvIII CAR-T cells by piggyBac transposon system, and detected its specific killing effect against SMMC7721 cells in vitro and in vivo. Results indicated that transduction efficiency of CAR reached 53.1%. Expression of CAR protein was verified by immunoblotting as a band of approximate 57KD. The killing effect of CAR-T cells against SMMC7721 was positively correlated with E/T ratio (E:T=5:1, 10:1, 20:1, 40:1), and exceeded 50% at 20:1 ratio. Significant increase in IFN-γ and TNF-α secretion were detected in the co-culture supernatant of CAR-T cells and SMMC7721, comparable to the level of exogenous EGFRvIII-expressing U87 cells. The killing activity and cytokine secretion were both dependent on the expression level of EGFRvIII in target cells. In HCC xenograft models, CAR-T cells could effectively suppress the growth of SMMC7721. In conclusion, EGFRvIII CAR-T cells demonstrated specific antitumor effect against SMMC7721 in vitro and in vivo, providing basis for immunotherapy of HCC in future clinical use.

Autologous non-human primate model for safety assessment of piggyBac transposon-mediated chimeric antigen receptor T cells on granulocyte-macrophage colony-stimulating factor receptor.

ObjectivesChimeric antigen receptor (CAR)‐T cell therapy redirected to specific antigens on tumor cells is a promising immunotherapy strategy for various cancers. Most target antigens are also expressed on normal tissues at varying levels, and therefore, a considerable challenge in the field is determining safety profiles, including life‐threatening off‐tumor and off‐target toxicities. The granulocyte–macrophage colony‐stimulating factor receptor (hGMR) is a promising target for CAR T‐cell therapy for a subset of acute myelocytic leukaemia, although it is also expressed on normal cells including monocytes, macrophages, CD34‐positive haematopoietic cells and vascular endothelial cells. hGMR and other immune‐related proteins are highly conserved between humans and cynomolgus macaques (Macaca fascicularis). Therefore, in this study, we engineered cynomolgus T cells to express CAR molecules redirected to hGMR by piggyBac (PB) transposon‐based gene transfer and adoptively transferred autologous hGMR‐CAR T cells into cynomolgus macaques.MethodsWe established PB‐mediated human GMR (hGMR)‐specific CAR T cells using cynomolgus peripheral blood mononuclear cells and transferred them into autologous individuals, and evaluated the potential toxicity related to hGMR‐CAR T cells.ResultshGMR‐CAR T cells did not exert overt organ toxicities such as bone marrow suppression, monocytopenia and vasculitis, although they recognised and killed cynomolgus monocytes and macrophages in vitro.ConclusionAlthough our model did not simulate a tumor‐bearing model, it supports the safety of hGMR‐CAR T cells and demonstrates the usefulness of a non‐human primate model to evaluate the safety of T‐cell products by assessing off‐tumor/off‐target toxicity before clinical trials.

Stable integration of an optimized inducible promoter system enables spatiotemporal control of gene expression throughout avian development.

ABSTRACTPrecisely altering gene expression is critical for understanding molecular processes of embryogenesis. Although some tools exist for transgene misexpression in developing chick embryos, we have refined and advanced them by simplifying and optimizing constructs for spatiotemporal control. To maintain expression over the entire course of embryonic development we use an enhanced piggyBac transposon system that efficiently integrates sequences into the host genome. We also incorporate a DNA targeting sequence to direct plasmid translocation into the nucleus and a D4Z4 insulator sequence to prevent epigenetic silencing. We designed these constructs to minimize their size and maximize cellular uptake, and to simplify usage by placing all of the integrating sequences on a single plasmid. Following electroporation of stage HH8.5 embryos, our tetracycline-inducible promoter construct produces robust transgene expression in the presence of doxycycline at any point during embryonic development in ovo or in culture. Moreover, expression levels can be modulated by titrating doxycycline concentrations and spatial control can be achieved using beads or gels. Thus, we have generated a novel, sensitive, tunable, and stable inducible-promoter system for high-resolution gene manipulation in vivo.

A Novel AAV-mediated Gene Delivery System Corrects CFTR Function in Pigs.

Cystic fibrosis is an autosomal-recessive disease that is caused by a mutant CFTR (cystic fibrosis transmembrane conductance regulator) gene and is characterized by chronic bacterial lung infections and inflammation. Complementation with functional CFTR normalizes anion transport across the airway surface. Adeno-associated virus (AAV) is a useful vector for gene therapy because of its low immunogenicity and ability to persist for months to years. However, because its episomal expression may decrease after cell division, readministration of the AAV vector may be required. To overcome this, we designed an integrating AAV-based CFTR-expressing vector, termed piggyBac (PB)/AAV, carrying CFTR flanked by the terminal repeats of the piggyBac transposon. With codelivery of the piggyBac transposase, PB/AAV can integrate into the host genome. Because of the packaging constraints of AAV, careful consideration was required to ensure that the vector would package and express its CFTR cDNA cargo. In this short-term study, PB/AAV-CFTR was aerosolized to the airways of CF pigs in the absence of the transposase. Two weeks later, transepithelial Cl- current was restored in freshly excised tracheal and bronchial tissue. Additionally, we observed an increase in tracheal airway surface liquid pH and bacterial killing in comparison with untreated CF pigs. Airway surface liquid from primary airway cells cultured from treated CF pigs exhibited increased pH correlating with decreased viscosity. Together, these results show that complementing CFTR in CF pigs with PB/AAV rescues the anion transport defect in a large-animal CF model. Delivery of this integrating viral vector system to airway progenitor cells could lead to persistent, life-long expression in vivo.

Genetic and genomic studies of pathogenic EXOSC2 mutations in the newly described disease SHRF implicate the autophagy pathway in disease pathogenesis.

Missense mutations in the RNA exosome component exosome component 2 (EXOSC2), also known as ribosomal RNA-processing protein 4 (RRP4), were recently identified in two unrelated families with a novel syndrome known as Short stature, Hearing loss, Retinitis pigmentosa and distinctive Facies (SHRF, #OMIM 617763). Little is known about the mechanism of the SHRF pathogenesis. Here we have studied the effect of mutations in EXOSC2/RRP4 in patient-derived lymphoblasts, clustered regularly interspaced short palindromic repeats (CRISPR)-generated mutant fetal keratinocytes and Drosophila. We determined that human EXOSC2 is an essential gene and that the pathogenic G198D mutation prevents binding to other RNA exosome components, resulting in protein and complex instability and altered expression and/or activities of critical genes, including those in the autophagy pathway. In parallel, we generated multiple CRISPR knockouts of the fly rrp4 gene. Using these flies, as well as rrp4 mutants with Piggy Bac (PBac) transposon insertion in the 3'UTR and RNAi flies, we determined that fly rrp4 was also essential, that fly rrp4 phenotypes could be rescued by wild-type human EXOSC2 but not the pathogenic form and that fly rrp4 is critical for eye development and maintenance, muscle ultrastructure and wing vein development. We found that overexpression of the transcription factor MITF was sufficient to rescue the small eye and adult lethal phenotypes caused by rrp4 inhibition. The autophagy genes ATG1 and ATG17, which are regulated by MITF, had similar effect. Pharmacological stimulation of autophagy with rapamycin also rescued the lethality caused by rrp4 inactivation. Our results implicate defective autophagy in SHRF pathogenesis and suggest therapeutic strategies.

PiggyBac Transposon-Based Immortalization of Human Deciduous Tooth Dental Pulp Cells with Multipotency and Non-Tumorigenic Potential.

We aimed to immortalize primarily isolated human deciduous tooth-derived dental pulp cells (HDDPCs) by transfection with piggyBac (PB)-based transposon vectors carrying E7 from human papilloma virus 16 or complementary DNA (cDNA) encoding human telomerase reverse transcriptase (hTERT). HDDPCs were co-transfected with pTrans (conferring PB transposase expression) + pT-pac (conferring puromycin acetyltransferase expression) + pT-tdTomato (conferring tdTomato cDNA expression) and pT-E7 (conferring E7 expression) or pTrans + pT-pac + pT-EGFP (conferring enhanced green fluorescent protein cDNA expression) + pT-hTERT (conferring hTERT expression). After six days, these cells were selected in medium containing 5 μg/mL puromycin for one day, and then cultured in normal medium allowing cell survival. All resultant colonies were harvested and propagated as a pool. Stemness and tumorigenic properties of the established cell lines (“MT_E7” for E7 and “MT_hTERT” for hTERT) with untransfected parental cells (MT) were examined. Both lines exhibited proliferation similar to that of MT, with alkaline phosphatase activity and stemness-specific factor expression. They displayed differentiation potential into multi-lineage cells with no tumorigenic property. Overall, we successfully obtained HDDPC-derived immortalized cell lines using a PB-based transfection system. The resultant and parental cells were indistinguishable. Thus, E7 and hTERT could immortalize HDDPCs without causing cancer-associated changes or altering phenotypic properties.

Genome Engineering Renal Epithelial Cells for Enhanced Volume Transport Function.

Bioengineering an implantable artificial kidney (IAK) will require renal epithelial cells capable of reabsorption of salt and water. We used genome engineering to modify cells for improved Na+/H+ exchange and H2O reabsorption. The non-viral piggyBac transposon system enables genome engineering cells to stably overexpress one or more transgenes simultaneously. We generated epitope-tagged human sodium hydrogen exchanger 3 (NHE3) and aquaporin-1 (AQP1) cDNA expressing piggyBac transposon vectors. Transgene expression was evaluated via western blot and immunofluorescence. Flow cytometry analysis was used to quantitate transporter expression in a library of genome engineered clones. Cell surface biotinylation was used evaluate surface protein localization. Blister formation assays were used to monitor cellular volumetric transport. piggyBac enabled stable transposon integration and overexpression of cumate-inducible NHE3 and/or constitutively expressing AQP1 in cultured renal (MDCK) epithelial cells. Cell surface delivery of NHE3 and AQP1 was confirmed using cell surface biotinylation assays. Flow cytometry of a library of MDCK clones revealed varying expression of AQP1 and NHE3. MDCK cells expressing AQP1 and cumate-inducible NHE3 demonstrated increased volumetric transport. Our results demonstrate that renal epithelial cells an be genome engineered for enhanced volumetric transport that will be needed for an IAK device. Our results lay the foundation for future studies of genome engineering human kidney cells for renal tubule cell therapy.

PiggyBac-engineered T cells expressing a glypican-3-specific chimeric antigen receptor show potent activities against hepatocellular carcinoma.

Glypican-3 (GPC3) is an attractive target for chimeric antigen receptor (CAR)-T cell therapy, as it is overexpressed in most hepatocellular carcinoma (HCC) tissues but shows restricted expression in healthy adult tissues. Herein, we generated GPC3-specific CAR-T cells for HCC therapy by electroporation with plasmid DNA encoding the piggyBac (PB) transposon and the hyperactive piggyBac transposase simultaneously instead of by commonly-used viral vectors. Our results demonstrated that GPC3CAR gene was efficiently integrated into the genome of T cells utilizing the PB transposon system. Upon stimulation with GPC3 antigen, GPC3CAR-T cells could be effectively activated, proliferate strongly and secrete high levels of cytokines. It also was demonstrated that GPC3CAR-T cells displayed potent cytotoxicity against GPC3-positive HCC cell lines in vitro by using real-time cell analyser (RTCA) system and the JuLI™ Stage Cell History Recorder. More importantly, in a Huh-7 xenograft mouse model, GPC3CAR-T cells significantly reduced the tumour burden companied with the secretion of high levels of IFN-γ. Moreover, T cells in mice treated with GPC3CAR-T cells could infiltrate into tumour tissues and persist as effector memory T cells (TEM). Overall, our study suggests that the use of PB system-based GPC3CAR-T cell therapy could be a promising clinical strategy for patients with HCC.

A simple and efficient method for the generation of a porcine alveolar macrophage cell line for high-efficiency Porcine reproductive and respiratory syndrome virus 2 infection

CD163 is a cellular receptor for Porcine reproductive and respiratory syndrome virus (PRRSV). Transgenic expression of CD163 can predispose a variety of PRRSV non-permissive cells to PRRSV infection. These resulting cells can then be used for PRRSV production and the study of PRRSV biology. The PiggyBac (PB) transposon is a non-viral, plasmid-based mobile genetic element that can be used for gene delivery into mammalian cells. In this study, a simple and efficient method for the transfection of the porcine CD163 transgene into an immortalized porcine alveolar macrophage cell line (3D4/21), a non-permissive cell line to PRRSV infection, by PB transposition was demonstrated. The resultant stably transformed 3D4/21/CD163 cells expressed CD163 constitutively and were shown to be fully permissive for PRRSV-2 strains and yielded an excess of 106 TCID50/mL of progeny virus. The PRRSV replicated more efficiently in the 3D4/21/CD163 cells than in Marc-145 cells, and the titers of the progeny PRRSV produced in the 3D4/21/CD163 cells were higher than those produced in Marc-145 cells. This simplified PB transposon-generated PRRSV-2 permissive 3D4/21/CD163 cell line could facilitate PRRSV production and accelerate the study of virus-host interactions in vitro.

584 Footprint-free gene mutation correction in induced pluripotent stem cell (iPSC) derived from recessive dystrophic epidermolysis bullosa (RDEB) using CRISPR/Cas9 and piggyBac transposon system

Recessive dystrophic epidermolysis bullosa is a monogenic skin blistering disorder caused by the mutations in the type VII collagen (COL7A1) gene. Combination of biological technologies, including induced pluripotent stem cells (iPSCs) and several gene-editing tools, allows us to realize gene and cell therapies for such inherited diseases. However, the methodology of gene and cell therapies must be consistently innovated for safety clinical use. In this study, we used CRISPR/Cas9 technology to correct the pathogenic mutation in RDEB-specific iPSCs, and piggyBac transposon system not to remain any residual gene fragments in the genome of iPSCs after correcting the mutation. We confirmed that mutation-corrected iPSCs (MC-iPSCs) had no genetic footprint and maintained “stemness”. When differentiating MC-iPSC into fibroblasts, the expression of type VII collagen was restored. Our study provided one of the safer approaches to establish gene and cell therapies for skin hereditary disorders for the future clinical use.

Efficient genome-wide first-generation phenotypic screening system in mice using the piggyBac transposon

Genome-wide phenotypic screens provide an unbiased way to identify genes involved in particular biological traits, and have been widely used in lower model organisms. However, cost and time have limited the utility of such screens to address biological and disease questions in mammals. Here we report a highly efficient piggyBac (PB) transposon-based first-generation (F1) dominant screening system in mice that enables an individual investigator to conduct a genome-wide phenotypic screen within a year with fewer than 300 cages. The PB screening system uses visually trackable transposons to induce both gain- and loss-of-function mutations and generates genome-wide distributed new insertions in more than 55% of F1 progeny. Using this system, we successfully conducted a pilot F1 screen and identified 5 growth retardation mutations. One of these mutants, a Six1/4 PB/+ mutant, revealed a role in milk intake behavior. The mutant animals exhibit abnormalities in nipple recognition and milk ingestion, as well as developmental defects in cranial nerves V, IX, and X. This PB F1 screening system offers individual laboratories unprecedented opportunities to conduct affordable genome-wide phenotypic screens for deciphering the genetic basis of mammalian biology and disease pathogenesis.

Baboon induced pluripotent stem cell generation by piggyBac transposition of reprogramming factors.

Clinical application of regenerative therapies using embryonic or induced pluripotent stem cells is within reach. Progress made during recent years has encouraged researchers to address remaining open questions in order to finally translate experimental cell replacement therapies into application in patients. To achieve this, studies in translationally relevant animal models are required to make the final step to the clinic. In this context, the baboon (Papio anubis) may represent a valuable nonhuman primate (NHP) model to test cell replacement therapies because of its close evolutionary relationship to humans and its large body size. In this study, we describe the reprogramming of adult baboon skin fibroblasts using the piggyBac transposon system. Via transposon-mediated overexpression of six reprogramming factors, we generated five baboon induced pluripotent stem cell (iPSC) lines. The iPSC lines were characterized with respect to alkaline phosphatase activity, pluripotency factor expression analysis, teratoma formation potential, and karyotype. Furthermore, after initial cocultivation with mouse embryonic fibroblasts, we were able to adapt iPSC lines to feeder-free conditions. In conclusion, we established a robust and efficient protocol for iPSC generation from adult baboon fibroblasts.

INVESTIGATING THE ROLE OF SORTILIN-RELATED RECEPTOR (SORL1/SORLA) IN HUMAN INDUCED PLURIPOTENT STEM CELL DERIVED NEURAL CELLS AS A THERAPEUTIC TARGET FOR ALZHEIMER’S DISEASE

Recent compelling genetic evidence has identified genes involved in endocytosis and vesicular trafficking including SORL1 as AD risk genes. Decreased expression of SORLA has been repeatedly observed in postmortem human AD brains especially in late onset sporadic AD (SAD) which is the most common form of AD. Eventually, several studies linked SORLA expression with production and deposition of the pathogenic protein β amyloid peptide which is one of the hallmarks of AD pathogenesis. However, most of these studies were done using mouse models and non-human cell lines and role of SORLA in amyloid β production in human neural cells remains unclear. SORL1 was overexpressed in clonal populations of human pluripotent stem cells (hiPSCs) using the piggybac transposon system. Control and SORL1overexpressed hiPSCs were then differentiated into neural cells. Amount of Aβ-40 and Aβ-42 released was measured using ELISA in hiPSC derived neural cells. Additionally, intra-cellular trafficking was examined using immunocytochemistry and cell biology assays. Specifically, to assess transport into lysosomes, pH sensitive fluorescent dye Phrodo conjugated with Aβ was used. Overexpression of SORL1 in hiPSC derived human neural cells resulted in decreased Aβ-40 and Aβ-42 released into media as measured by ELISA. This decrease can be caused by a possible increase in Aβ transport into lysosomes for degradation. In support of this idea, increased Aβ transport into lysosomes was observed in SORL1 overexpressed neural cells as compared to control cells using the Phrodo assay. Decreased Aβ release could also result from increased degradation of Amyloid precursor protein (APP). In support of this hypothesis, we showed increased colocalization of APP with a lysosomal marker, Lysosome associated membrane protein (LAMP1) using immunocytochemistry. Overall, these results suggest that overexpression of SORL1 could lead to decreased amyloid β production in part due to increased transport of Aβ and APP into lysosomes for degradation in human neural cells. Our findings reveal a previously unidentified role of SORL1 in hiPSC derived neural cells, information that may be useful in exploring not just SORLA, but other sorting receptors expressed in human neurons as therapeutic targets for AD.

The piggyBac-based double-inducible binary vector system: A novel universal platform for studying gene functions and interactions

Eukaryotic inducible overexpression systems, including Tet-On and mifepristone-inducible systems, have been widely used to study gene functions by reverse genetics. Among the transposon systems reported to date, the piggyBac transposon system is one of the most efficient in cultured mammalian cells. Here, we report a piggyBac-based double-inducible system that combined the advantages of previous systems. To create this system, the trans- and cis-elements of the Tet-On and mifepristone-inducible systems were cloned into a piggyBac-based trans-vector and cis-vector, respectively. The coding regions of two splicing variants of RUNX1, RUNX1a and RUNX1b, were inserted into the cis-vector to test its ability to express foreign genes along with fluorescent marker proteins. Transgenic 293 T cells were established, and the system was tested by inducing expression of foreign genes with DOX and/or mifepristone; GFP and/or mCherry were used as reporter genes. The system efficiently and stringently induced expression of GFP/mCherry and their co-expressed genes without significant mutual interference, as determined by qRT-PCR and Western blot. This piggyBac-based double-inducible system represents a new genetic tool for studying gene functions and interactions in vitro and in vivo in almost all organisms.

In Vivo Piggybac-Based Gene Delivery towards Murine Pancreatic Parenchyma Confers Sustained Expression of Gene of Interest.

The pancreas is a glandular organ that functions in the digestive system and endocrine system of vertebrates. The most common disorders involving the pancreas are diabetes, pancreatitis, and pancreatic cancer. In vivo gene delivery targeting the pancreas is important for preventing or curing such diseases and for exploring the biological function of genes involved in the pathogenesis of these diseases. Our previous experiments demonstrated that adult murine pancreatic cells can be efficiently transfected by exogenous plasmid DNA following intraparenchymal injection and subsequent in vivo electroporation using tweezer-type electrodes. Unfortunately, the induced gene expression was transient. Transposon-based gene delivery, such as that facilitated by piggyBac (PB), is known to confer stable integration of a gene of interest (GOI) into host chromosomes, resulting in sustained expression of the GOI. In this study, we investigated the use of the PB transposon system to achieve stable gene expression when transferred into murine pancreatic cells using the above-mentioned technique. Expression of the GOI (coding for fluorescent protein) continued for at least 1.5 months post-gene delivery. Splinkerette-PCR-based analysis revealed the presence of the consensus sequence TTAA at the junctional portion between host chromosomes and the transgenes; however, this was not observed in all samples. This plasmid-based PB transposon system enables constitutive expression of the GOI in pancreas for potential therapeutic and biological applications.

Modified CAR T cells targeting membrane-proximal epitope of mesothelin enhances the antitumor function against large solid tumor

Mesothelin (MSLN) is an attractive antigen for chimeric antigen receptor (CAR) T therapy and the epitope selection within MSLN is essential. In this study, we constructed two types of CARs targeting either region I of MSLN (meso1 CAR, also known as a membrane-distal region) or region III of MSLN (meso3 CAR, also known as a membrane-proximal region) using a modified piggyBac transposon system. We reported that, compared with meso1 CAR T cells, meso3 CAR T cells express higher levels of CD107α upon activation and produce increased levels of interleukin-2, TNF-α, and IFN-γ against multiple MSLN-expressing cancer cells in vitro. In a real-time cell analyzer system and a three-dimensional spheroid cancer cell model, we also demonstrated that meso3 CAR T cells display an enhanced killing effect compared with that of meso1 CAR T cells. More importantly, in a gastric cancer NSG mice model, meso3 CAR T cells mediated stronger antitumor responses than meso1 CAR T cells did. We further identified that meso3 CAR T cells can effectively inhibit the growth of large ovarian tumors in vivo. Collectively, our study provides evidences that meso3 CAR T-cell therapy performs as a better immunotherapy than meso1 CAR T-cell therapy in treating MSLN-positive solid tumors.

Characterization of soluble CD39 (SolCD39/NTPDase1) from PiggyBac nonviral system as a tool to control the nucleotides level.

Extracellular ATP (eATP) and its metabolites have emerged as key modulators of different diseases and comprise a complex pathway called purinergic signaling. An increased number of tools have been developed to study the role of nucleotides and nucleosides in cell proliferation and migration, influence on the immune system and tumor progression. These tools include receptor agonists/antagonists, engineered ectonucleotidases, interference RNAs and ectonucleotidase inhibitors that allow the control and quantification of nucleotide levels. NTPDase1 (also called apyrase, ecto-ATPase and CD39) is one of the main enzymes responsible for the hydrolysis of eATP, and purified enzymes, such as apyrase purified from potato, or engineered as soluble CD39 (SolCD39), have been widely used in in vitro and in vivo experiments. However, the commercial apyrase had its effects recently questioned and SolCD39 exhibits limitations, such as short half-life and need of high doses to reach the expected enzymatic activity. Therefore, this study investigated a non-viral method to improve the overexpression of SolCD39 and evaluated its impact on other enzymes of the purinergic system. Our data demonstrated that PiggyBac transposon system proved to be a fast and efficient method to generate cells stably expressing SolCD39, producing high amounts of the enzyme from a limited number of cells and with high hydrolytic activity. In addition, the soluble form of NTPDase1/CD39 did not alter the expression or catalytic activity of other enzymes from the purinergic system. Altogether, these findings set the groundwork for prospective studies on the function and therapeutic role of eATP and its metabolites in physiological and pathological conditions.

Matched sibling donor-derived piggybac CAR19 T cells induce remission of relapsed/refractory CD19+ malignancy following haematopoietic stem cell transplant

Background & Aim Aim The non-viral piggyBac transposon system reduces the complexity and expense of CAR T cell manufacture. piggyBac CAR19 T cells showed potent pre-clinical activity, so we now assess safety and clinical activity in a first-in-human study. Methods, Results & Conclusion Methods Eight patients (age 18-66 years, median 30; male n=7, female n=1) with relapsed/refractory CD19+ malignancies post HLA-matched sibling HSCT were treated with donor-derived T cells genetically modified using piggyBac to express a second-generation 4-1BB-based CAR19. The trial was conducted as an investigator-initiated study at a university hospital with local CAR19 T cell manufacture. CAR19 T cells were expanded ex vivo from 50 × 106 peripheral blood mononuclear cells, with CD19 stimulation and IL-15 support. Final products contained median 1.9 × 109 (range 0.8-2.5 × 109) total cells with 80.6% (39-93%) CAR T cells, and were generated in 15 (n=7) or 23 (n=1) days. Patients received up to 3 escalating doses of CAR19 T cells (10, 50 and 100 × 106/m2) following lymphodepleting cyclophosphamide and fludarabine (3 doses, n=5; 1 dose, n=3). Results Diagnoses were B-ALL (n=6), DLBCL (n=1), and Burkitt Lymphoma (n=1). Four patients each had a high or low burden of disease prior to treatment. The patient with Burkitt Lymphoma died of complications unrelated to CAR T cells 307 days with 3 doses. Toxicity included: cytokine release syndrome (n=3), B-cell aplasia with hypogammaglobulinaemia (n=7), prolonged cytopenias (n=6), bacterial infection (n=3) and exacerbation of chronic GVHD (n=1). No CAR T cell-related encephalopathy syndrome occurred. Conclusions Manufacture of piggyBac CAR19 T cells for clinical use is rapid, robust and uncomplicated, permitting application of this technology to multiple healthcare institutions. Early results suggest similar safety and activity to that of CAR19 T cells generated with viral vectors. The optimal CAR19 T cell dosing strategy remains to be determined.

Direct reprogramming to human nephron progenitor-like cells using inducible piggyBac transposon expression of SNAI2-EYA1-SIX1

All nephrons in the mammalian kidney arise from a transient nephron progenitor population that is lost close to the time of birth. The generation of new nephron progenitors and their maintenance in culture are central to the success of kidney regenerative strategies. Using a lentiviral screening approach, we previously generated a human induced nephron progenitor-like state invitro using a pool of six transcription factors. Here, we sought to develop a more efficient approach for direct reprogramming of human cells that could be applied invivo. PiggyBac transposons are a non-viral integrating gene delivery system that is suitable for invivo use and allows for simultaneous delivery of multiple genes. Using an inducible piggyBac transposon system, we optimized a protocol for the direct reprogramming of HK2 cells to induced nephron progenitor-like cells with expression of only 3 transcription factors (SNAI2, EYA1, and SIX1). Culture in conditions supportive of the nephron progenitor state further increased the expression of nephron progenitor genes. The refined protocol was then applied to primary human renal epithelial cells, which integrated into developing nephron structures invitro and invivo. Such inducible reprogramming to nephron progenitor-like cells could facilitate direct cellular reprogramming for kidney regeneration.

Production of Transgenic Porcine Embryos Reconstructed with Induced Pluripotent Stem-Like Cells Derived from Porcine Endogenous Factors Using piggyBac System.

The potential of induced pluripotent stem (iPS) cells, which have self-renewal ability and can differentiate into three germ layers, led us to hypothesize that iPS cells in pigs can be useful and suitable source for producing transgenic pigs. In this study, we generated iPS-like cells using doxycycline-inducible piggyBac (PB) expression vectors encoding porcine 4 transcription factors. After transfection, transfected cells were cultured until the formation of outgrowing colonies taking least of 7-10 days. The iPS-like cells demonstrated pluripotent characteristics such as self-renewal, high proliferation, expression of pluripotent markers, and aggregation ability. The embryo development through somatic cell nuclear transfer (SCNT), cleavage rate, and blastocyst formation rate did not show any significant differences. However, the total cell number of blastocysts was significantly increased with the established cell line. In conclusion, the iPS-like cell line, generated from porcine transcriptional factors using the PB transposon system, demonstrated pluripotency with the capacity for unlimited self-renewal, and could be used as donor cells to produce cloned embryos by SCNT. These cells will be suitable for gene modification and would contribute to the stability or safety of pig models in biomedical research.