Sleeping Beauty Transposition Research Articles

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

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

Sleeping Beauty Transposon Insertions into Nucleolar DNA by an Engineered Transposase Localized in the Nucleolus.

Transposons are nature's gene delivery vehicles that can be harnessed for experimental and therapeutic purposes. The Sleeping Beauty (SB) transposon shows efficient transposition and long-term transgene expression in human cells, and is currently under clinical development for gene therapy. SB transposition occurs into the human genome in a random manner, which carries a risk of potential genotoxic effects associated with transposon integration. Here, we evaluated an experimental strategy to manipulate SB's target site distribution by preferentially compartmentalizing the SB transposase to the nucleolus, which contains repetitive ribosomal RNA (rRNA) genes. We generated a fusion protein composed of the nucleolar protein nucleophosmin (B23) and the SB100X transposase, which was found to retain almost full transposition activity as compared to unfused transposase and to be predominantly localized to nucleoli in transfected human cells. Analysis of transposon integration sites generated by B23-SB100X revealed a significant enrichment into the p-arms of chromosomes containing nucleolus organizing regions (NORs), with preferential integration into the p13 and p11.2 cytobands directly neighboring the NORs. This bias in the integration pattern was accompanied by an enrichment of insertions into nucleolus-associated chromatin domains (NADs) at the periphery of nucleolar DNA and into lamina-associated domains (LADs). Finally, sub-nuclear targeting of the transposase resulted in preferential integration into chromosomal domains associated with the Upstream Binding Transcription Factor (UBTF) that plays a critical role in the transcription of 47S rDNA gene repeats of the NORs by RNA Pol I. Future modifications of this technology may allow the development of methods for specific gene insertion for precision genetic engineering.

Targeted delivery of a PD-1-blocking scFv by CD133-specific CAR-T cells using nonviral Sleeping Beauty transposition shows enhanced antitumour efficacy for advanced hepatocellular carcinoma

BackgroundCD133 is considered a marker for cancer stem cells (CSCs) in several types of tumours, including hepatocellular carcinoma (HCC). Chimeric antigen receptor-specific T (CAR-T) cells targeting CD133-positive CSCs have emerged as a tool for the clinical treatment of HCC, but immunogenicity, the high cost of clinical-grade recombinant viral vectors and potential insertional mutagenesis limit their clinical application.MethodsCD133-specific CAR-T cells secreting PD-1 blocking scFv (CD133 CAR-T and PD-1 s cells) were constructed using a sleeping beauty transposon system from minicircle technology, and the antitumour efficacy of CD133 CAR-T and PD-1 s cells was analysed in vitro and in vivo.ResultsA univariate analysis showed that CD133 expression in male patients at the late stage (II and III) was significantly associated with worse progression-free survival (PFS) (P = 0.0057) and overall survival (OS) (P = 0.015), and a multivariate analysis showed a trend toward worse OS (P = 0.041). Male patients with advanced HCC exhibited an approximately 20-fold higher PD-L1 combined positive score (CPS) compared with those with HCC at an early stage. We successfully generated CD133 CAR-T and PD-1 s cells that could secrete PD-1 blocking scFv based on a sleeping beauty system involving minicircle vectors. CD133 CAR-T and PD-1 s cells exhibited significant antitumour activity against HCC in vitro and in xenograft mouse models. Thus, CD133 CAR-T and PD-1 s cells may be a therapeutically tractable strategy for targeting CD133-positive CSCs in male patients with advanced HCC.ConclusionsOur study provides a nonviral strategy for constructing CAR-T cells that could also secrete checkpoint blockade inhibitors based on a Sleeping Beauty system from minicircle vectors and revealed a potential benefit of this strategy for male patients with advanced HCC and high CD133 expression (median immunohistochemistry score > 2.284).

HMGXB4 Targets Sleeping Beauty Transposition to Germinal Stem Cells.

Transposons are parasitic genetic elements that frequently hijack vital cellular processes of their host. HMGXB4 is a known Wnt signaling-regulating HMG-box protein, previously identified as a host-encoded factor of Sleeping Beauty (SB) transposition. Here, we show that HMGXB4 is predominantly maternally expressed, and marks both germinal progenitor and somatic stem cells. SB piggybacks HMGXB4 to activate transposase expression and target transposition to germinal stem cells, thereby potentiating heritable transposon insertions. The HMGXB4 promoter is located within an active chromatin domain, offering multiple looping possibilities with neighboring genomic regions. HMGXB4 is activated by ERK2/MAPK1, ELK1 transcription factors, coordinating pluripotency and self-renewal pathways, but suppressed by the KRAB-ZNF/TRIM28 epigenetic repression machinery, also known to regulate transposable elements. At the post-translational level, SUMOylation regulates HMGXB4, which modulates binding affinity to its protein interaction partners and controls its transcriptional activator function via nucleolar compartmentalization. When expressed, HMGXB4 can participate in nuclear-remodeling protein complexes and transactivate target gene expression in vertebrates. Our study highlights HMGXB4 as an evolutionarily conserved host-encoded factor that assists Tc1/Mariner transposons to target the germline, which was necessary for their fixation and may explain their abundance in vertebrate genomes.

779TiP Commencement of first-in-human phase I/II TCR-T clinical trial targeting shared tumor-specific hotspot mutations in solid tumors

Adoptive T-cell therapy is an emerging strategy for solid tumors. Cancer cells frequently harbor driver-mutations in KRAS, TP53, and EGFR genes that can be targeted by T-cell receptors (TCRs). These neoepitopes are presented on the tumor cell surface by human leukocyte antigen (HLA) molecules to TCRs. We have developed, using non-viral Sleeping Beauty transposition, a library of TCRs able to target KRAS, TP53 and EGFR mutations for the treatment of solid tumors.

Minicircles for CAR T Cell Production by Sleeping Beauty Transposition: A Technological Overview.

Development and application of chimeric antigen receptor (CAR) T cell therapy has led to a breakthrough in the treatment of hematologic malignancies. In 2017, the FDA approved the first commercialized CD19-specific CAR T cell products for treatment of patients with B-cell malignancies. This success increased the desire to broaden the availability of CAR T cells to a larger patient cohort with hematological but also solid tumors. A critical factor of CAR T cell production is the stable and efficient delivery of the CAR transgene into T cells. This gene transfer is conventionally achieved by viral vectors. However, viral gene transfer is not conducive to affordable, scalable, and timely manufacturing of CAR T cell products. Thus, there is a necessity for developing alternative nonviral engineering platforms, which are more cost-effective, less complex to handle and which provide the scalability requirement for a globally available therapy.One alternative method for engineering of T cells is the nonviral gene transfer by Sleeping Beauty (SB) transposition. Electroporation with two nucleic acids is sufficient to achieve stable CAR transfer into T cells. One of these vectors has to encode the gene of interest, which is the CAR , the second one a recombinase called SB transposase, the enzyme that catalyzes integration of the transgene into the host cell genome. As nucleic acids are easy to produce and handle SB gene transfer has the potential to provide scalability, cost-effectiveness, and feasibility for widespread use of CAR T cell therapies.Nevertheless, the electroporation of two large-size plasmid vectors into T cells leads to high T cell toxicity and low gene transfer rates and has hindered the prevalent clinical application of the SB system. To circumvent these limitations, conventional plasmid vectors can be replaced by minimal-size vectors called minicircles (MC ). MCs are DNA vectors that lack the plasmid backbone, which is relevant for propagation in bacteria, but has no function in a human cell. Thus, their size is drastically reduced compared to conventional plasmids. It has been demonstrated that MC-mediated SB CAR transposition into T cells enhances their viability and gene transfer rate enabling the production of therapeutic doses of CAR T cells. These improvements make CAR SB transposition from MC vectors a promising alternative for engineering of clinical grade CAR T cells.

Abstract 1515: Hotspot mutations in KRAS and TP53 targeted by TCR-T cells genetically modified with the Sleeping Beauty transposon/transposase system

Abstract Mutations in critical genes for cell survival and proliferation, e.g., KRAS and TP53, are found as clonal events in multiple tumor types of unrelated people likely due to their importance to the malignant phenotype. T cells recognize products of mutated genes, termed neoantigens, because they are expressed in the tumor but not in the normal tissues; thus, neoantigens are foreign entities from an immunological perspective. T-cell receptors (TCRs) with specificity to the neoantigen in the context of human leukocyte antigen (HLA) on the tumor cell surface can be isolated from the neoantigen-reactive T cell and potentially used for genetically-modified adoptive immunotherapy for any patient with matching mutation and HLA. The purpose of this study was to evaluate the ability of the non-viral Sleeping Beauty transposon/transposase gene transfer system to re-direct the specificity of T cells towards p53 and KRAS neoantigens and to characterize the resultant engineered TCR-T cell populations for specificity and function. Genes encoding the alpha and beta chains of p53 or KRAS neoantigen-specific TCRs were linked by a 2A ribosomal slip site linker and cloned into the clinical Sleeping Beauty transposon. These transposons were co-electroporated into donor peripheral blood leukocytes (PBL) with a DNA plasmid encoding the SB11 transposase and expanded in vitro. Logarithmic proliferation was observed and resulted in large numbers of highly pure TCR-T cells (>80% by introduced TCR expression). The TCR-T cells upregulated 41BB on the T-cell surface and secreted interferon-γ in response to antigen presenting cells with the appropriate HLA molecule pulsed with KRAS or p53 neoantigen peptides but not the cognate wild type peptide, confirming re-directed specificity to mutated KRAS or TP53 genes, respectively. Similarly, TCR-engineered T cells demonstrated cytolysis of tumor cells expressing the neoantigen and appropriate HLA restriction, suggesting that adoptive transfer of these TCR-T cells could mediate anti-tumor responses. In all, we demonstrated that multiple TCRs with unique specificities targeting recurrent p53 and KRAS substitutions in frequent HLA haplotypes could be stably expressed using Sleeping Beauty transposition to re-direct peripheral blood T cells towards tumor cells. Translation of these TCR-T cells into adoptive immunotherapy could result in safe and effective treatments for any cancer patient with matching HLA and KRAS or TP53 hotspot mutations. Citation Format: Ana B. Korngold, Lin-Kin Yong, Ming Zhang, Ugochi Ibekwe, Lenka V. Hurton, Yaoyao Shi, Julissa Simmons, Matthew R. Collinson-Pautz, Eleanor De Groot, Laurence J. Cooper, Drew C. Deniger. Hotspot mutations in KRAS and TP53 targeted by TCR-T cells genetically modified with the Sleeping Beauty transposon/transposase system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1515.

CARAMBA: a first-in-human clinical trial with SLAMF7 CAR-T cells prepared by virus-free Sleeping Beauty gene transfer to treat multiple myeloma

Clinical development of chimeric antigen receptor (CAR)-T-cell therapy has been enabled by advances in synthetic biology, genetic engineering, clinical-grade manufacturing, and complex logistics to distribute the drug product to treatment sites. A key ambition of the CARAMBA project is to provide clinical proof-of-concept for virus-free CAR gene transfer using advanced Sleeping Beauty (SB) transposon technology. SB transposition in CAR-T engineering is attractive due to the high rate of stable CAR gene transfer enabled by optimized hyperactive SB100X transposase and transposon combinations, encoded by mRNA and minicircle DNA, respectively, as preferred vector embodiments. This approach bears the potential to facilitate and expedite vector procurement, CAR-T manufacturing and distribution, and the promise to provide a safe, effective, and economically sustainable treatment. As an exemplary and novel target for SB-based CAR-T cells, the CARAMBA consortium has selected the SLAMF7 antigen in multiple myeloma. SLAMF7 CAR-T cells confer potent and consistent anti-myeloma activity in preclinical assays in vitro and in vivo. The CARAMBA clinical trial (Phase-I/IIA; EudraCT: 2019-001264-30) investigates the feasibility, safety, and anti-myeloma efficacy of autologous SLAMF7 CAR-T cells. CARAMBA is the first clinical trial with virus-free CAR-T cells in Europe, and the first clinical trial that uses advanced SB technology worldwide.

Jumping Ahead with Sleeping Beauty: Mechanistic Insights into Cut-and-Paste Transposition.

Sleeping Beauty (SB) is a transposon system that has been widely used as a genetic engineering tool. Central to the development of any transposon as a research tool is the ability to integrate a foreign piece of DNA into the cellular genome. Driven by the need for efficient transposon-based gene vector systems, extensive studies have largely elucidated the molecular actors and actions taking place during SB transposition. Close transposon relatives and other recombination enzymes, including retroviral integrases, have served as useful models to infer functional information relevant to SB. Recently obtained structural data on the SB transposase enable a direct insight into the workings of this enzyme. These efforts cumulatively allowed the development of novel variants of SB that offer advanced possibilities for genetic engineering due to their hyperactivity, integration deficiency, or targeting capacity. However, many aspects of the process of transposition remain poorly understood and require further investigation. We anticipate that continued investigations into the structure–function relationships of SB transposition will enable the development of new generations of transposition-based vector systems, thereby facilitating the use of SB in preclinical studies and clinical trials.

RNA-guided retargeting of Sleeping Beauty transposition in human cells.

An ideal tool for gene therapy would enable efficient gene integration at predetermined sites in the human genome. Here we demonstrate biased genome-wide integration of the Sleeping Beauty (SB) transposon by combining it with components of the CRISPR/Cas9 system. We provide proof-of-concept that it is possible to influence the target site selection of SB by fusing it to a catalytically inactive Cas9 (dCas9) and by providing a single guide RNA (sgRNA) against the human Alu retrotransposon. Enrichment of transposon integrations was dependent on the sgRNA, and occurred in an asymmetric pattern with a bias towards sites in a relatively narrow, 300 bp window downstream of the sgRNA targets. Our data indicate that the targeting mechanism specified by CRISPR/Cas9 forces integration into genomic regions that are otherwise poor targets for SB transposition. Future modifications of this technology may allow the development of methods for specific gene insertion for precision genetic engineering.

H20Y Mutation Stabilizes Structure of the Primary DNA Recognition Domain of the Sleeping Beauty Transposase

The ability of DNA transposons to jump from one DNA site or molecule to another is exploited in functional genomics, genetic engineering, and human gene therapy. Sleeping Beauty (SB) is the most widely used DNA transposon in genetic applications. The onset of SB transposition is the recognition of the transposon DNA by the PAI subdomain of SB transposase. Previously, we have determined that the PAI subdomain remains mostly unstructured at experimental conditions that mimic physiologic. We have further established that only the folded conformation of the PAI subdomain is capable of binding to the transposon DNA. Here, we computationally identify and experimentally prove, using NMR and CD spectroscopy, several structure-stabilizing mutation in the PAI subdomain, including H20Y. We also show that introducing H20Y mutation increases DNA binding of the PAI subdomain. Our data suggest that introducing structure-stabilizing H20Y mutation into the SB transposase could improve the efficiency of SB transposition.

203 Genome Modifications by Sleeping Beauty Transposition and CRISPR/Cas9 to Improve Cow Milk Composition for Human Consumption

Genome manipulation of cattle represents a powerful tool to increase the nutritional value and reduce allergenicity of cow milk for human consumption. This could be accomplished by improving the amount of polyunsaturated fatty acids (ω-3 and ω-6) and simultaneously abolishing β-lactoglobulin (BLG), a potent allergen for predisposed humans. The aim of this study was to introduce the sequence for a desaturase construct (mFAT-2, from C. elegans), which is able to catalyse the synthesis of ω-3 and ω-6 fatty acids, into the bovine genome by Sleeping Beauty (SB) transposition, and simultaneously knocking out the bovine β-lactoglubulin gene using CRISPR/Cas9 system. The sgRNA (AAGTGCCTCCTGCTTGCCC) targeted to BLG exon 1 was synthesised as an oligo linker and cloned into the px459-Cas9. The mutation activity of the designed sgRNA at the target locus was determined by T7 endonuclease assay I (T7EI) mismatch detection assay. Briefly, bovine fetal fibroblasts (BFF) were seeded at 0.5 × 105 cells per well of a 24-well plate in triplicate, when the cells reached 80% confluence (12–24 h), cultures were transfected with 1 μg of px459-Cas9::BLG plasmid co-expressing Cas9 and sgRNA using polyethylenimine reagent (PEI; 3 ng μL−1). After 3 days of puromycin selection, genomic DNA from transfected cells were extracted and the sequence of interest was PCR-amplified and digested by T7EI restriction enzyme. Digestion products showed a mutation efficiency at the target locus of 29%. Subsequently, we chemically cotransfected 0.5 × 105 BFF with 0.5 μg of knockout vector (px459-Cas9::BLG) and 0.5 μg of SB plasmids (carrying mFAT-2 cDNA for mammary gland-specific expression) using 3 ng μL−1 PEI in triplicate. At 48 h post-transfection, cell cultures were subjected to 3 days of puromycin and 21 days of neomycin selection. PCR analysis of antibiotic resistant colonies revealed the presence of mFAT-2 transgene in almost 70% of the analysed cells lines. Genotyping of BLG exon 1 was performed by direct sequencing of PCR amplicons using primers flanking the target site. Despite the appreciable gene mutation activity of the sgRNA sequence previously determined by T7EI assay (29%), none of the cell lines analysed showed modification in the BLG target locus. We speculate that the SB vector might have disrupted the activity of targeting vector. We are currently performing additional experiments to accomplish gene addition (mFAT) and gene knockout (BGL) in one step using these highly efficient and precise transgenic tools. Genetically modified cells will be used as nuclear donor to produce transgenic cows by somatic cells nuclear transfer. The financial support of CONICET, UNRC and FONCYT is gratefully acknowledged.

NMR solution structure of the RED subdomain of the Sleeping Beauty transposase.

DNA transposons can be employed for stable gene transfer in vertebrates. The Sleeping Beauty (SB) DNA transposon has been recently adapted for human application and is being evaluated in clinical trials, however its molecular mechanism is not clear. SB transposition is catalyzed by the transposase enzyme, which is a multi-domain protein containing the catalytic and the DNA-binding domains. The DNA-binding domain of the SB transposase contains two structurally independent subdomains, PAI and RED. Recently, the structures of the catalytic domain and the PAI subdomain have been determined, however no structural information on the RED subdomain and its interactions with DNA has been available. Here, we used NMR spectroscopy to determine the solution structure of the RED subdomain and characterize its interactions with the transposon DNA.

Enhanced CAR T-cell engineering using non-viral Sleeping Beauty transposition from minicircle vectors.

Immunotherapy with T cell modified with gamma-retroviral or lentiviral (LV) vectors to express a chimeric antigen receptor (CAR) has shown remarkable efficacy in clinical trials. However, the potential for insertional mutagenesis and genotoxicity of viral vectors is a safety concern, and their cost and regulatory demands a roadblock for rapid and broad clinical translation. Here, we demonstrate that CAR T cells can be engineered through non-viral Sleeping Beauty (SB) transposition of CAR genes from minimalistic DNA vectors called minicircles (MCs). We analyzed genomic distribution of SB and LV integrations and show that a significantly higher proportion of MC-derived CAR transposons compared with LV integrants had occurred outside of highly expressed and cancer-related genes into genomic safe harbor loci that are not expected to cause mutagenesis or genotoxicity. CD19-CAR T cells engineered with our enhanced SB approach conferred potent reactivity in vitro and eradicated lymphoma in a xenograft model in vivo. Intriguingly, electroporation of SB MCs is substantially more effective and less toxic compared with conventional plasmids, and enables cost-effective rapid preparation of therapeutic CAR T-cell doses. This approach sets a new standard in advanced cellular and gene therapy and will accelerate and increase the availability of CAR T-cell therapy to treat hematologic malignancies.

Detection of Sleeping Beauty transposition in the genome of host cells by non-radioactive Southern blot analysis

The Sleeping Beauty transposon (SB-Tn) system is being used widely as a DNA vector for the delivery of therapeutic transgenes, as well as a tool for the insertional mutagenesis in animal models. In order to accurately assess the insertional potential and properties related to the integration of SB it is essential to determine the copy number of SB-Tn in the host genome. Recently developed SB100X transposase has demonstrated an integration rate that was much higher than the original SB10 and that of other versions of hyperactive SB transposases, such as HSB3 or HSB17. In this study, we have constructed a series of SB vectors carrying either a DsRed or a human β-globin transgene that was encompassed by cHS4 insulator elements, and containing the SB100X transposase gene outside the SB-Tn unit within the same vector in cis configuration. These SB-Tn constructs were introduced into the K-562 erythroid cell line, and their presence in the genomes of host cells was analyzed by Southern blot analysis using non-radioactive probes. Many copies of SB-Tn insertions were detected in host cells regardless of transgene sequences or the presence of cHS4 insulator elements. Interestingly, the size difference of 2.4 kb between insulated SB and non-insulated controls did not reflect the proportional difference in copy numbers of inserted SB-Tns. We then attempted methylation-sensitive Southern blots to assess the potential influence of cHS4 insulator elements on the epigenetic modification of SB-Tn. Our results indicated that SB100X was able to integrate at multiple sites with the number of SB-Tn copies larger than 6 kb in size. In addition, the non-radioactive Southern blot protocols developed here will be useful to detect integrated SB-Tn copies in any mammalian cell type.

Imaging of Sleeping Beauty-Modified CD19-Specific T Cells Expressing HSV1-Thymidine Kinase by Positron Emission Tomography.

We have incorporated a positron emission tomography (PET) functionality in T cells expressing a CD19-specific chimeric antigen receptor (CAR) to non-invasively monitor the adoptively transferred cells. We engineered T cells to express CD19-specific CAR, firefly luciferase (ffLuc), and herpes simplex virus type-1 thymidine kinase (TK) using the non-viral-based Sleeping Beauty (SB) transposon/transposase system adapted for human application. Electroporated primary T cells were propagated on CD19+ artificial antigen-presenting cells. After 4weeks, 90% of cultured cells exhibited specific killing of CD19+ targets in vitro, could be ablated by ganciclovir, and were detected in vivo by bioluminescent imaging and PET following injection of 2'-deoxy-2'-[18F]fluoro-5-ethyl-1-β-D-arabinofuranosyl-uracil ([18F]FEAU). This is the first report demonstrating the use of SB transposition to generate T cells which may be detected using PET laying the foundation for imaging the distribution and trafficking of T cells in patients treated for B cell malignancies.

73. Enhanced Engineering of Chimeric Antigen Receptor (CAR)-Modified T Cells Using Non-Viral Sleeping Beauty Transposition from Minicircle Vectors

Adoptive therapy with T cells that were modified with gamma-retroviral and lentiviral (LV) vectors to express a CD19-specific chimeric antigen receptor (CAR) has shown remarkable efficacy in pilot clinical trials. However, there are persistent concerns with the use of viral vectors with regard to safety, as well as the cost and scale of vector production required for making CAR T-cell therapy available to patients on a global level. In this study, we have refined non-viral Sleeping Beauty (SB) transposition to provide an effective and broadly applicable gene-transfer strategy with superior safety profile. We demonstrate that SB transposition of CAR transgenes from minicircle (MC) DNA vectors enables dramatically improved transposition rates compared to conventional plasmids. MC-derived CD19-CAR transposons display a highly favorable integration profile and confer stable CAR expression that permits potent anti-tumor functions and rapid manufacture of CAR T-cell products.We prepared MC transposon donor vectors encoding a CD19-CAR or eGFP from corresponding parental pT2 plasmids through site specific recombination, and a MC encoding SB100X transposase. Each MC contained only the promotor and gene of interest, and was devoid of antibiotic resistance gene and bacterial origin of replication. We then performed transfections into CD8+ and CD4+ T cells of healthy donors (n=7) and analyzed transposition rate and stability that could be accomplished when CD19-CAR transposon and SB100X transposase were encoded by MCs vs. equimolar amounts of corresponding conventional plasmids. In each donor, we achieved a significantly higher transposition rate with MCs (mean 49.8% on day 14 post transfection) compared to plasmids (mean 12.8%; 4.4-fold difference; p<0,001). Expression of the CD19-CAR was stable over multiple rounds of expansion and several weeks in culture. Importantly, MC transfection was not only more effective, but also substantially less toxic compared to plasmids and on average, a 6-fold higher yield of CAR T cells could be obtained within 14 days of culture without the need for feeder cell expansion. In functional experiments, CD19-CAR T cells modified with our enhanced MC-based SB transposition strategy were equally effective as LV transduced CD19-CAR T cells, and exerted high levels of specific cytolytic activity, cytokine production including IFN-g and IL-2, and productive proliferation after stimulation with CD19+ lymphoma. Moreover, a single administration of SB-modified CD19-CAR T cells leads to complete eradication of systemic lymphoma in a murine xenograft model (NSG/Raji-ffLuc), and was at least equivalently potent as LV transduced CD19-CAR T cells prepared from the same donor. To address safety, we determined the gene copy number (n=5, range 3-8 in CD8+ T cells) and performed a comprehensive genomic insertion site analysis. The data show a close-to-random integration profile of MC-derived CD19-CAR transposons, without preference for highly expressed or cancer related genes. Intriguingly, a significantly higher proportion of SB integrations had occurred in genomic safe harbors compared to LV integrations (7-fold difference; p<0,001), close to the perfect score expected for random integration. In conclusion, we demonstrate the potential to manufacture CAR T cells using virus-free SB-mediated transposition from MC DNA vectors. The superior safety profile, high level stable transposition rate and ease-of-handling MC vectors position our novel approach to become a preferred gene-transfer strategy in advanced cellular and gene-therapy.

Stable, Nonviral Expression of Mutated Tumor Neoantigen-specific T-cell Receptors Using the Sleeping Beauty Transposon/Transposase System.

Neoantigens unique to each patient's tumor can be recognized by autologous T cells through their T-cell receptor (TCR) but the low frequency and/or terminal differentiation of mutation-specific T cells in tumors can limit their utility as adoptive T-cell therapies. Transfer of TCR genes into younger T cells from peripheral blood with a high proliferative potential could obviate this problem. We generated a rapid, cost-effective strategy to genetically engineer cancer patient T cells with TCRs using the clinical Sleeping Beauty transposon/transposase system. Patient-specific TCRs reactive against HLA-A*0201-restriced neoantigens AHNAK(S2580F) or ERBB2(H473Y) or the HLA-DQB*0601-restricted neoantigen ERBB2IP(E805G) were assembled with murine constant chains and cloned into Sleeping Beauty transposons. Patient peripheral blood lymphocytes were coelectroporated with SB11 transposase and Sleeping Beauty transposon, and transposed T cells were enriched by sorting on murine TCRβ (mTCRβ) expression. Rapid expansion of mTCRβ(+) T cells with irradiated allogeneic peripheral blood lymphocytes feeders, OKT3, interleukin-2 (IL-2), IL-15, and IL-21 resulted in a preponderance of effector (CD27(-)CD45RA(-)) and less-differentiated (CD27(+)CD45RA(+)) T cells. Transposed T cells specifically mounted a polyfunctional response against cognate mutated neoantigens and tumor cell lines. Thus, Sleeping Beauty transposition of mutation-specific TCRs can facilitate the use of personalized T-cell therapy targeting unique neoantigens.

Minicircle-Based Engineering of Chimeric Antigen Receptor (CAR) T Cells.

Plasmid DNA is being used as a pharmaceutical agent in vaccination, as well as a basic substance and starting material in gene and cell therapy, and viral vector production. Since the uncontrolled expression of backbone sequences present in such plasmids and the dissemination of antibiotic resistance genes may have profound detrimental effects, an important goal in vector development was to produce supercoiled DNA lacking bacterial backbone sequences: Minicircle (MC) DNA. The Sleeping Beauty (SB) transposon system is a non-viral gene delivery platform enabling a close-to-random profile of genomic integration. In combination, the MC platform greatly enhances SB transposition and transgene integration resulting in higher numbers of stably modified target cells. We have recently developed a strategy for MC-based SB transposition of chimeric antigen receptor (CAR) transgenes that enable improved transposition rates compared to conventional plasmids and rapid manufacturing of therapeutic CAR T cell doses (Monjezi et al. 2016). This advance enables manufacturing CAR T cells in a virus-free process that relies on SB-mediated transposition from MC DNA to accomplish gene-transfer. Advantages of this approach include a strong safety profile due to the nature of the MC itself and the genomic insertion pattern of MC-derived CAR transposons. In addition, stable transposition and high-level CAR transgene expression, as well as easy and reproducible handling, make MCs a preferred vector source for gene-transfer in advanced cellular and gene therapy. In this chapter, we will review our experience in MC-based CAR T cell engineering and discuss our recent advances in MC manufacturing to accelerate both pre-clinical and clinical implementation.

Serum-based tracking of de novo initiated liver cancer progression reveals early immunoregulation and response to therapy

Background & AimsLiver inflammatory diseases associated with cancer promoting somatic oncogene mutations are increasing in frequency. Preclinical cancer models that allow for the study of early tumor progression are often protracted, which limits the experimental study parameters due to time and expense. Here we report a robust inexpensive approach using Sleeping Beauty transposition (SBT) delivery of oncogenes along with Gaussia Luciferase expression vector GLuc, to assess de novo liver tumor progression, as well as the detection of innate immune responses or responses induced by therapeutic intervention. MethodsTracking de novo liver tumor progression with GLuc was demonstrated in models of hepatocellular carcinoma (HCC) or adenoma (HCA) initiated by hydrodynamic delivery of SBT oncogenes. ResultsRising serum luciferase levels correlated directly with increasing liver tumor burden and eventual morbidity. Early detection of hepatocyte apoptosis from mice with MET+CAT transfected hepatocytes was associated with a transient delay in HCC growth mediated by a CD8+ T-cell response against transformed hepatocytes. Furthermore, mice that lack B cells or macrophages had an increase in TUNEL+ hepatocytes following liver MET transfection demonstrating that these cells provide protection from MET-induced hepatocyte apoptosis. Treatment with IL-18+IL-12 of mice displaying established HCC decreased tumor burden which was associated with decreased levels of serum luciferase. ConclusionsHydrodynamic delivery of the SBT vector GLuc to hepatocytes serves as a simple blood-based approach for real-time tracking of pathologically distinct types of liver cancer. This revealed tumor-induced immunologic responses and was beneficial in monitoring the efficacy of therapeutic interventions.