Hyperactive Sleeping Beauty Transposase Research Articles

Construction and Quantitative Evaluation of a Tissue-Specific Sleeping Beauty by EDL2-Specific Transposase Expression in Esophageal Squamous Carcinoma Cell Line KYSE-30.

Gene delivery to esophageal tissue could provide novel treatments for diseases, such as cancer. The Sleeping Beauty (SB) transposon system, as a natural and non-viral tool, is efficient at transferring transgene into the human genome for human cell genetic engineering. The plasmid-based SB transposon can insert into chromosomes through an accurate recombinase-mediated mechanism, providing long-term expression of transgene integrated into the target cells. In this study, we aimed to investigate the activity of ED-L2 tissue-specific promoter that was engineered from the Epstein-Barr Virus (EBV) and combined with the hyperactive SB100X transposase to achieve the stable expression of T2-Onc3 transposon in esophageal squamous epithelial cells. Here we constructed an SB transposon-based plasmid system to obtain the stable expression of transposon upon introduction of a hyperactive SB transposase under the control of tissue-specific ED-L2 promoter via the lipid-based delivery method in the cultured esophageal squamous cell carcinoma cells. Among established human and mouse cell lines, the (ED-L2)-SB100X transposase was active only in human esophageal stratified squamous epithelial and differentiated keratinocytes derived from skin (KYSE-30 and HaCaT cell lines), where it revealed high promoter activity. Data offered that the 782bp sequence of ED-L2 promoter has a key role in its activity in vitro. The (ED-L2)-SB100X transposase mediated stable integration of T2-Onc3 in KYSE-30 cells, thereby providing further evidence of the tissue specificity of ED-L2 promoter. The KYSE-30 cells modified with the SB system integrate on average 187 copies of the T2-Onc3 transposon in its genome. In aggregate, the (ED-L2)-SB100X transposase can be efficiently applied for the tissue-specific stable expression of a transgene in human KYSE-30 cells using SB transposon.

Persistent Control of SIV Infection in Rhesus Macaques By Expressing a Highly Potent SIV Decoy Receptor after In Vivo HSC Transduction

Despite the success achieved by antiretroviral HIV-1 therapies, long-term and repeated drug administration is associated with toxicity, virus evasion and high cost. We aim to develop a gene therapy approach for persistent control against HIV-1 infection by delivering the transgene expressing a decoy receptor (eCD4-Ig) to hematopoietic stem cells (HSCs) by in vivo transduction. In this approach, HSCs are mobilized from the bone marrow into the peripheral blood stream and transduced with intravenously injected virus vectors. We use an integrating, helper-dependent adenovirus (HDAd5/35++) vector system that targets human CD46, a receptor that is abundantly expressed on primitive HSCs. Transgene integration is achieved by a hyperactive Sleeping Beauty transposase and transgene marking in peripheral blood cells can be increased by in vivo selection. The efficacy and safety of our in vivo HSC transduction/selection strategy has been previously demonstrated for the treatment of b-hemoglobinopathies, hemophilia A, and cancer in murine disease models. In non-human primates, we showed efficient transgene (g-globin) expression in peripheral red blood cells using this strategy. eCD4-Ig functions like a neutralizing antibody and shows broad neutralization spectrum against HIV-1, HIV-2 and SIV isolates. We have designed and produced an HDAd5/35++ vector expressing rhesus eCD4-Ig (rh-eCD4-Ig) from a constitutive and highly active EF1a promoter. In CD46-transgenic mice, over 50µg/mL eCD4-Ig in serum was measured after in vivo transduction and selection, with no obvious adverse events observed. Neutralization assay with serum samples showed that the produced eCD4-Ig effectively inhibited HIV-1 and SIV infection. We then performed studies in a rhesus macaque. After in vivo HSC transduction/selection of a rhesus macaque, eCD4-Ig serum levels were stable at 20-30 mg/ml. In vitro SIVmac239 neutralization assays using week 13 serum and recombinant eCD4-Ig protein determined that the IC50 of eCD4-Ig in rhesus serum is 1.0 mg/ml. This implies that the serum eCD4-Ig concentration at the time of SIVmac239 challenge was ~25-fold higher than the IC50. High-level eCD4-Ig expression had no clinical or hematological side effects. The first SIVmac239 challenge (20pg) was given on June 22 nd. Increasing rechallenge doses are currently injected monthly. So far, the viral load measured by quantitative RT-PCR is below detection limit. The animal is without symptoms and has normal lymphocyte/subset counts. Our study demonstrates an in vivo HSC transduction approach for potential long-term control of HIV-1 infection. DisclosuresKiem: VOR Biopharma: Consultancy; Ensoma Inc.: Consultancy, Current holder of individual stocks in a privately-held company; Homology Medicines: Consultancy. Lieber: Ensoma: Research Funding.

In Vivo HSC Transduction and Selection Results in Long-Term, High-Level Expression of Human Gamma Globin in Peripheral Blood Erythrocytes of Mice

Current protocols for HSC gene therapy, involving the transplantation of ex vivo lentivirus vector-transduced HSCs into myeloablated recipients, are complex and not without risk for the patient. We have developed a new approach for in vivo gene delivery into HSCs that does not require myeloablation and HSC transplantation. It involves injections of G-CSF/AMD3100 to mobilize HSCs from the bone marrow into the peripheral blood stream and the intravenous injection of an integrating, helper-dependent adenovirus (HDAd5/35++) vector system. Transgene integration is achieved (in a random pattern) using a hyperactive Sleeping Beauty transposase (SB100x). We demonstrated in adequate mouse models, using GFP as a transgene, that primitive HSCs transduced in the periphery home back to the bone marrow where they persist and stably express GFP long-term (Blood 128:2206 (2016). To achieve high level transgene marking in differentiated peripheral blood cells, we combined our in vivo HSC transduction approach with in vivo selection of transduced HSCs using the mgtmP140K mutant and low dose O6BG/BCNU treatment. Here were tested the potential of this approach for the therapy of hemoglobinopathies. Our HDAd5/35++ vector contained the human-gamma globin gene under the control of a 6kb version of the beta-globin LCR (containing HS1 to HS4 and the beta globin promoter) and the mgmtP140K gene under the control of the PGK promoter. Mobilized immunocompetent hCD46 transgenic mice were injected IV with the integrating vector system and subjected to three rounds of treatment with low-dose O6BG/BCNU followed by immunosuppression to avoid immune responses against the human transgene product. The procedure was safe and resulted in stable human gamma globin expression in >95% of erythrocytes in the peripheral blood of in vivo transduced cells mice. Human gamma globin levels were 100% of endogenous mouse beta globin levels in erythrocytes measured by HPLC. Analysis of bone marrow at week 16 after in vivo transduction showed that >60% of bone marrow LSK cells contained the integrated transgene cassette and that the beta globin LCR efficiently restricted gamma globin expression to erythroid cells. After transplantation of unsorted, lineage-depleted bone marrow cells into lethally irradiated recipients, long-term gamma-globin expression at levels seen in the primary in vivo treated mice was demonstrated. The efficacy of our approach achieved in this study indicates that it would be curative in thalassemia models or patients. Our new approach avoids the collection of HSCs, their transduction ex vivo, and subsequent transplantation into myeloablated recipients. It therefore could greatly simplify HSC gene therapy of common diseases. DisclosuresKiem:Rocket Pharmaceuticals: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

Update on the Sleeping Beauty transposon system: current state-of-the-art & remaining challenges

Zoltán Ivics received his PhD in molecular biology in 1994. After postoctoral studies at the University of Minnesota in the USA and the Netherlands Cancer Institute, he was appointed as a research group leader at the Max Delbrück Center for Molecular Medicine in Berlin, Germany. He was appointed as Head of Division at the Paul Ehrlich Institute in Langen, Germany, in 2011. Prof. Ivics’ major scientific achievement is the molecular reconstruction of the Sleeping Beauty transposon and development of technologies based on Sleeping Beauty gene transfer for a wide array of applications involving genetic engineering of cells. Prof. Ivics has published >130 papers in peer reviewed journals, with a total Impact Factor of >800 and >7000 citations (h-index: 44), and is co-inventor on 12 patents. Since 2000 his research efforts were supported by >20 research grants from the German Research Foundation, the German Ministry of Education and Research, the European Commission, and the Volkswagen Foundation. He received recognition of the ‘Molecule of the Year’ in 2009 for developing a hyperactive Sleeping Beauty transposase that opened the door for clinical applications. He is a current member of the Board of the European Society of Gene and Cell Therapy (ESGCT), member of the Board of the German Society of Gene Therapy and member of the committee for ‘Clinical trials and regulatory affairs’ of the German Stem Cell Network. Prof. Ivics organized several international conferences, including the Annual Congress of the ESGCT in Berlin in 2017. He is an Elected Member of the Academia Europaea since 2018.

High-level protein production in erythroid cells derived from in vivo transduced hematopoietic stem cells

AbstractWe developed an in vivo hematopoietic stem cell (HSC) transduction approach that involves HSC mobilization from the bone marrow into the peripheral bloodstream and the IV injection of an integrating, helper-dependent adenovirus (HDAd5/35++) vector system. HDAd5/35++ vectors target human CD46, a receptor that is abundantly expressed on primitive HSCs. Transgene integration is achieved by a hyperactive Sleeping Beauty transposase (SB100x) and transgene marking in peripheral blood cells can be increased by in vivo selection. Here we directed transgene expression to HSC-derived erythroid cells using β-globin regulatory elements. We hypothesized that the abundance and systemic distribution of erythroid cells can be harnessed for high-level production of therapeutic proteins. We first demonstrated that our approach allowed for sustained, erythroid-lineage specific GFP expression and accumulation of GFP protein in erythrocytes. Furthermore, after in vivo HSC transduction/selection in hCD46-transgenic mice, we demonstrated stable supraphysiological plasma concentrations of a bioengineered human factor VIII, termed ET3. High-level ET3 production in erythroid cells did not affect erythropoiesis. A phenotypic correction of bleeding was observed after in vivo HSC transduction of hCD46+/+/F8−/− hemophilia A mice despite high plasma anti-ET3 antibody titers. This suggests that ET3 levels were high enough to provide sufficient noninhibited ET3 systemically and/or locally (in blood clots) to control bleeding. In addition to its relevance for hemophilia A gene therapy, our approach has implications for the therapy of other inherited or acquired diseases that require high levels of therapeutic proteins in the blood circulation.

Thalassemia Gene Therapy By In Vivo Transduction of Mobilized Hematopoietic Stem Cells (HSCs) with an Integrating Hybrid Adenovirus Vector System

To overcome the cost and complexity of current thalassemia ex vivogene therapy protocols, we developed a minimally invasive and readily translatable approach for in vivo HSC gene delivery which abrogates the need for HSC leukapheresis, CD34+ cell selection, ex vivo HSC culture, myeloablation and ultimately, transplantation. Our approach involves HSC mobilization with G-CSF/AMD3100 andintravenous injections of a hybrid vector system consisting ofa CD46-targeting, helper-dependent adenoviral vector and the hyperactive Sleeping Beauty transposase (SB100x) that mediates integration of thevector-encoded γ-globin and mgmtP140K genes. Pretreatment with glucocorticoids before virus injectionsis used to blockthe release of pro-inflammatory cytokines andimmunosuppression is applied in order to avoid responses against human g-globin- and MGMT protein-expressing cells. We tested our approach in a mouse model recapitulating the phenotypeof human β-thalassemia intermedia (Hbbth-3/hCD46++ mice). At week 8 post transduction, hCD46+/+/Hbbth-3 mice expressed HbF in 31.2±2.7% of circulating erythrocytes. Due to a significant drop in HbF expression by week 16 (11.9±3.0%), a 4-dose O6BG/BCNU treatment was administeredin order to in vivo select forgene corrected hematopoietic progenitors, thus recovering the HbF expression in76.0±5.7% of the circulating erythrocytes, by week 29 post in vivo transduction. With an average vector copy number of 1.4/cell, the human γ-globin to mouse α-globin expression was ~10% by HPLC and the human γ-globin to mouse β-globin mRNA ratio ~10%, by qRT-PCR. Hematological parameters (RBCs, Ht, Hb, MCV, RDW, Reticulocytes) at week 29 post in vivo transduction, were significantly improved over baseline or were indistinguishable from normal values, suggesting near to complete phenotypic correction. Treated mice showed significant reduction of spleen size, extramedullary erythropoiesis and parenchymal hemosiderosis. After secondary transplantation and without in vivo selection, more than 90% of donor-derived erythrocytes (CD46+) were g-globin-positive, up to 20 weeks post-transplant. Safety was demonstrated by the good tolerability of treatment, the absence of alterations in hematopoiesis, the normal colony-forming potential of bone marrow cells and the random integration pattern of our vector system. Overall, we present a simplified platform for gene therapy of thalassemia, which can serve as a cost-efficient and “portable” approach to make gene therapy accessible even to resource-poor regions where thalassemia major is endemic but only minimally complex strategies could be adopted. DisclosuresNo relevant conflicts of interest to declare.

106. A Sleeping Beauty Transposase System in the Context of HD-Ad5/35++ Vectors Achieves Stable In Vivo Transduction of Hematopoietic Stem Cells in Mouse Models

Hematopoietic stem cells (HSCs) are important targets for the gene therapy of infectious diseases, genetic disorders, and cancer. Our aim is to transduce HSCs in vivo after mobilization from the bone marrow and intravenous injection of a gene transfer vector. We employed helper-dependent adenovirus vector containing Ad35 fiber mutants with enhanced affinity to CD46 (HD-Ad5/35++) for gene transfer into HSCs. HD-Ad5/35++ vectors efficiently transduced human HSCs and human CD46-transgenic mouse HSCs in vitro. To trigger chromosomal transgene integration we utilized the hyperactive Sleeping Beauty transposase system (SB100x). The system consists of two vectors; the transposon vector carrying a GFP gene and the SB100x vector that mediates integration of the GFP cassette into a TA dinucleotide of the genomic DNA. As a limitation, this system requires the co-infection of the same target cell with two vectors to achieve stable transduction. This is exemplified in a study with two Ad5/35++ reporter vectors, expressing either GFP or mCherry. Human CD46tg mice were mobilized with GSCF/AMD3100 and intravenously injected with either vector alone or the vector combination. Reporter gene expression in bone marrow localized HSCs was analyzed 3 days later. While 2% of lineage depleted cells expressed either GFP or mCherry only 0.25% expressed both. In spite of these limitations, we tested whether the co-injection of a HD-Ad5/35++ -GFP transposon vector and a HD-Ad5/35++ SB100x vector, supplying a Sleeping Beauty transposase in trans into mobilized mice would lead to stable HSC transduction. Twelve weeks post-injection, we detected stable GFP expression in bone marrow HSCs in the range of 1 to 2%. GFP integration through SB100X was confirmed by genome-wide sequencing of integration sites. Our data indicate that HD-Ad5/35++ vectors preferentially target primitive HSCs and that this increases the chance of co-infection with both vectors. This is supported by a comparison of GFP marking in HSCs and more differentiated marrow and blood cells four weeks after in vivo transduction. While in the HSC-containing LSK fraction of the bone marrow the marking rate was on average 7.7%, marking rates for total mononuclear cells of the bone marrow and peripheral blood were only 2.5% and 0.3%, respectively. The preferential HSC targeting in hCD46tg and humanized mice can be explained by higher CD46 levels on primitive HSCs compared to more differentiated cells in the bone marrow and the dependence of Ad5/35 infection efficiency (at low MOIs) on CD46 density on the cell surface.

A High-Capacity Adenoviral Hybrid Vector System Utilizing the Hyperactive Sleeping Beauty Transposase SB100X for Enhanced Integration.

For efficient delivery of required genetic elements we utilized high-capacity adenoviral vectors in the past allowing high transgene capacities of up to 36 kb. Previously we explored the hyperactive Sleeping Beauty (SB) transposase (HSB5) for somatic integration from the high-capacity adenoviral vectors genome. To further improve this hybrid vector system we hypothesized that the previously described hyperactive SB transposase SB100X will result in significantly improved efficacies after transduction of target cells. Plasmid based delivery of the SB100X system revealed significantly increased integration efficiencies compared with the previously published hyperactive SB transposase HSB5. After optimizing experimental setups for high-capacity adenoviral vectors-based delivery of the SB100X system we observed up to eightfold and 100-fold increased integration efficiencies compared with the previously published hyperactive SB transposase HSB5 and the inactive transposase mSB, respectively. Furthermore, transposon copy numbers per cell were doubled with SB100X compared with HSB5 when using the identical multiplicity of infection. We believe that this improved hybrid vector system represents a valuable tool for achieving stabilized transgene expression in cycling cells and for treatment of numerous genetic disorders. Especially for in vivo approaches this improved adenoviral hybrid vector system will be advantageous because it may potentially allow reduction of the applied viral dose.

Transcriptionally regulated and nontoxic delivery of the hyperactive Sleeping Beauty Transposase

The Sleeping Beauty (SB) transposase and, in particular, its hyperactive variant SB100X raises increasing interest for gene therapy application, including genome modification and, more recently, induced pluripotent stem cells (iPS) reprogramming. The documented cytotoxicity of the transposase, when constitutively expressed by an integrating retroviral vector (iRV), has been circumvented by the transient delivery of SB100X using retroviral mRNA transfer. In this study, we developed an alternative, safe, and efficient transposase delivery system based on a tetracycline-ON regulated expression cassette and the rtTA2S-M2 transactivator gene transiently delivered by integration-defective lentiviral vectors (IDLVs). Compared with iRV-mediated delivery, expression of tetracycline-induced SB100X delivered by an IDLV results in more efficient integration of a GFP transposon and reduced toxicity. Tightly regulated expression and reactivation of the transposase was achieved in HeLa cells as wells as in human primary keratinocytes. Based on these properties, the regulated transposase-IDLV vectors may represent a valuable tool for genetic engineering and therapeutic gene transfer.

Pre-Emptive Donor Lymphocyte Infusion with CD19-Directed, CAR-Modified T Cells Infused after Allogeneic Hematopoietic Cell Transplantation for Patients with Advanced CD19+ Malignancies

Background: Allogeneic hematopoietic cell transplantation (HCT) can be curative in a subset of patients with advanced lymphoid malignancies but relapse remains a major reason for treatment failure. Donor-derived, non-specific lymphocyte infusions (DLI) can confer an immune anti-malignancy effect but can be complicated by graft-versus-host-disease (GVHD). Chimeric antigen receptor (CAR)-modified T cells directed toward CD19 have demonstrated dramatic efficacy in patients with refractory ALL and NHL. However, responses are often associated with life-threatening cytokine release syndrome. Aim: We hypothesized that infusing CAR-modified, CD19-specific T-cells after HCT as a directed DLI would be associated with a low rate of GVHD, better disease control, and a less severe cytokine release syndrome since administered in a minimal disease state. Methods: We employed a non-viral gene transfer using the Sleeping Beauty (SB) transposon/transposase system to stably express a CD19-specific CAR (designated CD19RCD28 that activates via CD3z & CD28) in donor-derived T cells for patients with advanced CD19+ lymphoid malignancies. T-cells were electroporated using a Nucleofector device to synchronously introduce two DNA plasmids coding for SB transposon (CD19RCD28) and hyperactive SB transposase (SB11). T-cells stably expressing the CAR were retrieved over 28 days of co-culture by recursive additions of g-irradiated activating and propagating cells (AaPC) in presence of soluble recombinant interleukin (IL)-2 and IL-21. The AaPC were derived from K562 cells and genetically modified to co-express CD19 as well as the co-stimulatory molecules CD86, CD137L, and a membrane-bound version of IL-15. Results: To date, we have successfully treated 21 patients with median age 36 years (range 21-62) with advanced CD19+ ALL (n=18) or NHL (n=3); 10 patients had active disease at time of HCT. Donor-derived CAR+ T cells (HLA-matched sibling n=10; 1 Ag mismatched sibling n=1; haplo family n=8; cord blood n=2) were infused at a median 64 days (range 42-91 days) following HCT to prevent disease progression. Transplant preparative regimens were myeloablative, busulfan-based (n=10) or reduced intensity, fludarabine-based (n=11). All patients were maintained on GVHD prophylaxis at time of CAR T-cell infusion with tacrolimus, plus mycophenolate mofeteil for cord, plus post-HCT cyclophosphamide for haplo donors. The starting CAR+ T-cell dose was 106 (n=7), escalated to 107 (n=6), 5x107 (n=5), and currently at 108 (n=3) modified T cells/m2 (based on recipient body surface area). Patients have not demonstrated any acute or late toxicity to CAR+ T cell infusions. Three patients developed acute grades 2-4 GVHD (liver n=1, upper GI n=1, skin=1) which was within the expected range after allogeneic HCT alone. Of note, the rate of CMV reactivation after CAR T cell infusion was 24% vs. 41 % previously reported for our patients without CAR T cell infusion (Wilhelm et al. J Oncol Parm Practice, 2014, 20:257). Nineteen patients have had at least 30 days follow-up post CAR T-cell infusion and are evaluable for disease progression. Forty-eight percent of patients (n=10) remain alive and in complete remission (CR) at median 5.2 months (range 0-21.3 months) following CAR T cell infusion. Importantly, among 8 patients who received haplo-HCT and CAR, 7 remain in remission at median 4.2 months. Conclusion: We demonstrate that infusing donor-derived CD19-specific CAR+ T cells, using the SB and AaPC platform, in the adjuvant HCT setting as pre-emptive DLI may provide an effective and safe approach for maintaining remission in patients at high risk for relapse. Graft-vs-host disease did not appear increased by administration of the donor derived CAR-T cells. Furthermore, the add-back of allogeneic T cells appears to have contributed to immune reconstitution and control of opportunistic viral infection. Disclosures Huls: Intrexon and Ziopharm: Employment, Equity Ownership. Singh:Intrexon and Ziopharm: Equity Ownership, Patents & Royalties. Olivares:Intrexon and Ziopharm: Equity Ownership, Patents & Royalties. Su:Ziopharm and Intrexon: Employment. Figliola:Intrexon and Ziopharm: Equity Ownership, Patents & Royalties. Kumar:Ziopharm and Intrexon: Equity Ownership. Jena:Ziopharm Oncology: Equity Ownership, Patents & Royalties: Potential roylaties (Patent submitted); Intrexon: Equity Ownership, Patents & Royalties: Potential royalties (Patent submitted). Ang:Intrexon and Ziopharm: Equity Ownership. Lee:Intrexon: Equity Ownership; Cyto-Sen: Equity Ownership; Ziopharm: Equity Ownership.

Transposon-Based, Targeted Ex Vivo Gene Therapy to Treat Age-Related Macular Degeneration (TargetAMD).

Transposon-Based, Targeted Ex Vivo Gene Therapy to Treat Age-Related Macular Degeneration (TargetAMD).

Adoptive Therapy Using Sleeping Beauty Gene Transfer System and Artificial Antigen Presenting Cells to Manufacture T Cells Expressing CD19-Specific Chimeric Antigen Receptor

Objectives: T cells can be genetically modified ex vivo to redirect specificity upon expression of a chimeric antigen receptor (CAR) that recognizes tumor-associated antigen (TAA) independent of human leukocyte antigen. We employ non-viral gene transfer using the Sleeping Beauty (SB) transposon/transposase system to stably express a 2nd generation CD19-specific CAR- (designated CD19RCD28 that activates via CD3z/CD28) in patient (pt)- or donor-derived T cells for patients with advanced B-cell malignancies.Methods: T cells were electroporated using a Nucleofector device to synchronously introduce two DNA plasmids coding for SB transposon (CD19RCD28) and hyperactive SB transposase (SB11). T cells stably expressing the CAR were retrieved over 28 days of co-culture by recursive additions of designer g-irradiated activating and propagating cells (AaPC) in presence of soluble recombinant interleukin (IL)-2 and IL-21. The aAPC were derived from K562 cells and genetically modified to co-express the TAA CD19 as well as the co-stimulatory molecules CD86, CD137L, and a membrane-bound protein of IL-15. The dual platforms of the SB system and aAPC are illustrated in figure below.Results: To date we have successfully manufactured product for 42 pts with multiply-relapsed ALL (n=19), NHL (n=17), or CLL (n=5) on 4 investigator-initiated trials at MD Anderson Cancer Center to administer thawed pt- and donor-derived CD19-specific T cells as planned infusions in the adjuvant setting after autologous (n=5), allogeneic (n=21) or umbilical cord (n=4) hematopoietic cell transplantation (HCT), or for the treatment of active disease (n=12). Each clinical-grade T-cell product was subjected to a battery of in-process and final release testing. Adjuvant trials: Twelve pts have been infused with donor-derived CAR+ T cells following allogeneic HCT, including 2 pts with cord blood-derived T cells (ALL, n=10; NHL, n=2), beginning at a dose of 106 and escalating to 5x107 modified T cells/m2. Three pts, all with ALL, remain alive and in remission at median 5 months following T cell infusion. Five pts with NHL have been treated with pt-derived modified T cells following autologous HCT at a dose of 5x108 T cells/m2, and 4 pts remain in remission at median 12 months following T-cell infusions. Relapse trials: Thirteen pts have been treated for active disease (ALL, n=8; NHL, n=3; CLL, n=2) with pt or donor-derived (if prior allo-HCT) modified T cells at doses 106-5x107/m2, and 3 remain alive and in remission at median 3 months following T-cell infusions. No acute or late toxicities, including excess GVHD, have been noted.Conclusion: We report the first human application of the SB and AaPC systems to genetically modify clinical-grade cells. Furthermore, infusing CD19-specific CAR+ T cells in the adjuvant HCT setting and thus targeting minimal residual disease may provide an effective and safe approach for maintaining remission in pts at high risk for relapse.Next steps: The SB system serves as a nimble and cost-effective platform for genetic engineering of T cells. We are implementing next-generation clinical T-cell trials targeting ROR1, releasing T cells for infusion within days after electro-transfer of SB DNA plasmid coding for CAR and mRNA coding for transposase, and infusing T cells modified with CAR designs with improved therapeutic potential. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Correction: Integration Profile and Safety of an Adenovirus Hybrid-Vector Utilizing Hyperactive Sleeping Beauty Transposase for Somatic Integration

Correction: Integration Profile and Safety of an Adenovirus Hybrid-Vector Utilizing Hyperactive Sleeping Beauty Transposase for Somatic Integration

Adoptive Immunotherapy Following Umbilical Cord Blood Transplantation Using The Sleeping Beauty System and Artificial Antigen Presenting Cells To Generate Donor-Derived T Cells Expressing a CD19-Specific Chimeric Antigen Receptor

▪ BackgroundThe ability to transplant across HLA disparities makes allogeneic umbilical cord blood (UCB) an attractive graft source for hematopoietic stem-cell transplantation (HSCT). Disease relapse remains a limitation, and adoptive transfer of tumor-specific T cells post UCB HSCT has not been feasible due to the functionally naïve CB T cells, and the small size as well as anonymity of the donor. We report a new approach to non-viral gene transfer using the Sleeping Beauty (SB) transposon/transposase system to stably express a 2nd generation CD19-specific chimeric antigen receptor (CAR, designated CD19RCD28) on UCB-derived T cells manufactured in compliance with current good manufacturing practice (cGMP). MethodsAfter thawed UCB units are washed for clinical infusion 5% to 10% of cells are used to generate CAR+ T cells. The mononuclear cells are electroporated using a Nucleofector device to synchronously introduce two DNA plasmids coding for SB transposon (CD19RCD28) and hyperactive SB transposase (SB11). T cells stably expressing the CAR are retrieved over 28 days of co-culture by recursive additions of g-irradiated artificial antigen presenting cells (aAPC) in presence of soluble recombinant interleukin (IL)-2 and IL-21. The aAPC (designated clone #4) were derived from K562 cells and genetically modified to co-express the CD19 as well as the co-stimulatory molecules CD86, CD137L, and a membrane-bound protein of IL-15. Enrolled patients on our phase I trial receive two UCB units, thus two genetically modified T-cell products are made for each patient. We infuse thawed donor-derived CD19-specific CAR+ T cells from the dominant CB unit based on peripheral blood chimerism on days 40-100 post transplant in the adjuvant setting after double UCB HSCT ResultsTo date we have successfully manufactured 8 products for 4 patients (ALL n=3, NHL=1) enrolled on trial. The median number of T cells in the starting CB aliquot was 8.6x106 (range, 2.5x106 to 54.8x106) with final modified T cell count at median 3x109 (range,1.7x108 to 4.1x1010) at time of cryopreservation days 28-32. In the final product, the median CD19-CAR+ cell purity by flow was 88% (range, 81.9% to 95.8%). The modified T cell product consisted of median 97.3% CD3+, 2.7 CD3-/CD56+ cells. All of the products exhibited CD19-specific killing by chromium assay as illustrated (Figure). Each clinical-grade T-cell product was subjected to a battery of in-process testing to complement release testing. One patient with ALL has been infused to date with a T cell dose of 106T cells/m2 and no toxicity has been observed. The patient remains alive and in continued molecular remission at 111 days post HSCT. ConclusionWe combined the SB system and aAPC-mediated propagation of T cells to successfully manufacture disease-specific T cells from small aliquots of UCB used to restore hematopoiesis. Importantly, this approach allows us to employ adoptive therapy to enhance the graft-versus-tumor effect in UCB HSCT as an approach to improve overall survival for these recipients. Accrual to the trial continues and updated results will be presented at the meeting. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

First Clinical Trials Employing Sleeping Beauty Gene Transfer System and Artificial Antigen Presenting Cells To Generate and Infuse T Cells Expressing CD19-Specific Chimeric Antigen Receptor

▪ BackgroundT cells can be genetically modified ex vivo to redirect specificity upon enforced expression of a chimeric antigen receptor (CAR) that recognizes tumor-associated antigen (TAA) independent of human leukocyte antigen. We report a new approach to non-viral gene transfer using the Sleeping Beauty (SB) transposon/transposase system to stably express a 2nd generation CD19-specific CAR- (designated CD19RCD28 that activates via CD3z/CD28) in autologous and allogeneic T cells manufactured in compliance with current good manufacturing practice (cGMP) for Phase I/II trials. MethodsT cells were electroporated using a Nucleofector device to synchronously introduce DNA plasmids coding for SB transposon (CD19RCD28) and hyperactive SB transposase (SB11). T cells stably expressing the CAR were retrieved over 28 days of co-culture by recursive additions of g-irradiated artificial antigen presenting cells (aAPC) in presence of soluble recombinant interleukin (IL)-2 and IL-21. The aAPC (designated clone #4) were derived from K562 cells and genetically modified to co-express the TAA CD19 as well as the co-stimulatory molecules CD86, CD137L, and a membrane-bound protein of IL-15. The dual platforms of the SB system and aAPC are illustrated in figure below. ResultsTo date we have enrolled and manufactured product for 25 patients with multiply-relapsed ALL (n=12) or B-cell lymphoma (n=13) on three investigator-initiated trials at MD Anderson Cancer Center to administer thawed patient- and donor-derived CD19-specific T cells as planned infusions in the adjuvant setting after autologous (n=7), allogeneic adult (n=14) or umbilical cord (n=4) hematopoietic stem-cell transplantation (HSCT). Each clinical-grade T-cell product was subjected to a battery of in-process testing to complement release testing under CLIA. Currently, five patients have been infused with the CAR+ T cells following allogeneic HSCT, including one patient with cord blood-derived T cells (ALL, n=4; NHL, n=1), beginning at a dose of 106 and escalating to 107 modified T cells/m2. Three patients treated at the first dose level of 106 T cells/m2 have progressed; the patient treated at the next dose level with 107 T cells/m2 remains in remission at 5 months following HSCT. Assessment for response too early for patient treated with UCB T cells. Four patients with non-Hodgkin's lymphoma have been treated with patient-derived modified T cells following autologous HSCT at a dose of 5x107 T cells/m2, and all patients remain in remission at 3 months following HSCT. No acute or late toxicities have been noted to date. PCR testing for persistence of CAR-modified T cells is underway. ConclusionWe report the first human application of the SB and aAPC systems to genetically modify clinical-grade cells. Importantly, infusing CD19-specific CAR+ T cells in the adjuvant HSCT setting and thus targeting minimal residual disease is feasible and safe, and may provide an effective approach for maintaining remission in patients with high risk, CD19+ lymphoid malignancies. Clinical data is accruing and will be updated at the meeting. This nimble manufacturing approach can be readily modified in a cost-effective manner to improve the availability, persistence and therapeutic potential of genetically modified T cells, as well as target tumor–associated antigens other than CD19. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Integration Profile and Safety of an Adenovirus Hybrid-Vector Utilizing Hyperactive Sleeping Beauty Transposase for Somatic Integration

We recently developed adenovirus/transposase hybrid-vectors utilizing the previously described hyperactive Sleeping Beauty (SB) transposase HSB5 for somatic integration and we could show stabilized transgene expression in mice and a canine model for hemophilia B. However, the safety profile of these hybrid-vectors with respect to vector dose and genotoxicity remains to be investigated. Herein, we evaluated this hybrid-vector system in C57Bl/6 mice with escalating vector dose settings. We found that in all mice which received the hyperactive SB transposase, transgene expression levels were stabilized in a dose-dependent manner and that the highest vector dose was accompanied by fatalities in mice. To analyze potential genotoxic side-effects due to somatic integration into host chromosomes, we performed a genome-wide integration site analysis using linker-mediated PCR (LM-PCR) and linear amplification-mediated PCR (LAM-PCR). Analysis of genomic DNA samples obtained from HSB5 treated female and male mice revealed a total of 1327 unique transposition events. Overall the chromosomal distribution pattern was close-to-random and we observed a random integration profile with respect to integration into gene and non-gene areas. Notably, when using the LM-PCR protocol, 27 extra-chromosomal integration events were identified, most likely caused by transposon excision and subsequent transposition into the delivered adenoviral vector genome. In total, this study provides a careful evaluation of the safety profile of adenovirus/Sleeping Beauty transposase hybrid-vectors. The obtained information will be useful when designing future preclinical studies utilizing hybrid-vectors in small and large animal models.

Efficient Sleeping Beauty DNA Transposition From DNA Minicircles

DNA transposon-based vectors have emerged as new potential delivery tools in therapeutic gene transfer. Such vectors are now showing promise in hematopoietic stem cells and primary human T cells, and clinical trials with transposon-engineered cells are on the way. However, the use of plasmid DNA as a carrier of the vector raises safety concerns due to the undesirable administration of bacterial sequences. To optimize vectors based on the Sleeping Beauty (SB) DNA transposon for clinical use, we examine here SB transposition from DNA minicircles (MCs) devoid of the bacterial plasmid backbone. Potent DNA transposition, directed by the hyperactive SB100X transposase, is demonstrated from MC donors, and the stable transfection rate is significantly enhanced by expressing the SB100X transposase from MCs. The stable transfection rate is inversely related to the size of circular donor, suggesting that a MC-based SB transposition system benefits primarily from an increased cellular uptake and/or enhanced expression which can be observed with DNA MCs. DNA transposon and transposase MCs are easily produced, are favorable in size, do not carry irrelevant DNA, and are robust substrates for DNA transposition. In accordance, DNA MCs should become a standard source of DNA transposons not only in therapeutic settings but also in the daily use of the SB system.

Manipulating piggyBac Transposon Chromosomal Integration Site Selection in Human Cells

The ability to direct gene delivery to a user-defined chromosomal location would greatly improve gene transfer applications. The piggyBac transposon system is a nonviral gene transfer system proven effective in a variety of cells and tissues, including human cells. We fused a highly site-specific synthetic zinc-finger DNA-binding domain (ZFP) to the N-terminus of the piggyBac transposase and evaluated site-directed genomic integration in human cells. Chimeric ZFP-piggyBac transposase exhibited robust gene transfer activity, targeted binding to a cognate endogenous chromosomal ZFP site in the human genome, and site-directed transposon integration into an episomal plasmid target containing a single ZFP site in human cells. We evaluated the ability of ZFP-piggyBac to direct gene integration into an engineered chromosomal ZFP target site in the human genome and consistently observed a higher degree of ZFP-piggyBac site-directed genomic integration when compared to native piggyBac. Chromatin immunoprecipitation (ChIP) experiments revealed binding of native piggyBac to our engineered TTAA-containing chromosomal target which supported integration, but not a TTAA-deficient chromosomal target which lacked integration. Our results offer insight into the requirements for using a chimeric zinc finger-piggyBac transposase to direct integration into a user-defined chromosomal location.

The hyperactive Sleeping Beauty transposase SB100X improves the genetic modification of T cells to express a chimeric antigen receptor

Sleeping Beauty (SB3) transposon and transposase constitute a DNA plasmid system used for therapeutic human cell genetic engineering. Here we report a comparison of SB100X, a newly developed hyperactive SB transposase, to a previous generation SB11 transposase to achieve stable expression of a CD19-specific chimeric antigen receptor (CAR3) in primary human T cells. The electro-transfer of SB100X expressed from a DNA plasmid or as an introduced mRNA species had superior transposase activity in T cells based on the measurement of excision circles released after transposition and emergence of CAR expression on T cells selectively propagated upon CD19+ artificial antigen-presenting cells. Given that T cells modified with SB100X and SB11 integrate on average one copy of the CAR transposon in each T-cell genome, the improved transposition mediated by SB100X apparently leads to an augmented founder effect of electroporated T cells with durable integration of CAR. In aggregate, SB100X improves SB transposition in primary human T cells and can be titrated with an SB transposon plasmid to improve the generation of CD19-specific CAR+ T cells.

Hyperactive Sleeping Beauty Transposase Enables Persistent Phenotypic Correction in Mice and a Canine Model for Hemophilia B

Sleeping Beauty (SB) transposase enables somatic integration of exogenous DNA in mammalian cells, but potency as a gene transfer vector especially in large mammals has been lacking. Herein, we show that hyperactive transposase system delivered by high-capacity adenoviral vectors (HC-AdVs) can result in somatic integration of a canine factor IX (cFIX) expression-cassette in canine liver, facilitating stabilized transgene expression and persistent haemostatic correction of canine hemophilia B with negligible toxicity. We observed stabilized cFIX expression levels during rapid cell cycling in mice and phenotypic correction of the bleeding diathesis in hemophilia B dogs for up to 960 days. In contrast, systemic administration of an inactive transposase system resulted in rapid loss of transgene expression and transient phenotypic correction. Notably, in dogs a higher viral dose of the active SB transposase system resulted into transient phenotypic correction accompanied by transient increase of liver enzymes. Molecular analysis of liver samples revealed SB-mediated integration and provide evidence that transgene expression was derived mainly from integrated vector forms. Demonstrating that a viral vector system can deliver clinically relevant levels of a therapeutic protein in a large animal model of human disease paves a new path toward the possible cure of genetic diseases.