Stable gene transfer and expression in human primary T cells by the Sleeping Beauty transposon system

Huang, Xin; Wilber, Andrew; Bao, Lei; Tuong, Dong; Tolar, Jakub; Orchard, Paul J.; Levine, Bruce L.; June, Carl H.; McIvor, R. Scott; Blazar, Bruce R.; Zhou, Xianzheng

doi:10.1182/blood-2005-05-2133

Cited by 98 publications

(76 citation statements)

References 53 publications

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“…We note that in these studies a transposase of intermediate activity (SB11; ref. 35) was used, rather than the more active SB100X transposase that may direct multiple integrations of transposons into individual cells (68,69). Another advantage of our approach is that we avoided the expense and inconvenience associated with steady-state apheresis, as the electroporation and propagation of T cells was accomplished from PB obtained by venipuncture.…”

Section: Discussionmentioning

confidence: 99%

Phase I trials using Sleeping Beauty to generate CD19-specific CAR T cells

Kebriaei¹,

Singh

Huls

et al. 2016

Journal of Clinical Investigation

425

390

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BACKGROUND.T cells expressing antigen-specific chimeric antigen receptors (CARs) improve outcomes for CD19-expressing B cell malignancies. We evaluated a human application of T cells that were genetically modified using the Sleeping Beauty (SB) transposon/transposase system to express a CD19-specific CAR. METHODS.T cells were genetically modified using DNA plasmids from the SB platform to stably express a second-generation CD19-specific CAR and selectively propagated ex vivo with activating and propagating cells (AaPCs) and cytokines. Twenty-six patients with advanced non-Hodgkin lymphoma and acute lymphoblastic leukemia safely underwent hematopoietic stem cell transplantation (HSCT) and infusion of CAR T cells as adjuvant therapy in the autologous (n = 7) or allogeneic settings (n = 19).RESULTS. SB-mediated genetic transposition and stimulation resulted in 2,200-to 2,500-fold ex vivo expansion of genetically modified T cells, with 84% CAR expression, and without integration hotspots. Following autologous HSCT, the 30-month progression-free and overall survivals were 83% and 100%, respectively. After allogeneic HSCT, the respective 12-month rates were 53% and 63%. No acute or late toxicities and no exacerbation of graft-versus-host disease were observed. Despite a low antigen burden and unsupportive recipient cytokine environment, CAR T cells persisted for an average of 201 days for autologous recipients and 51 days for allogeneic recipients.CONCLUSIONS. CD19-specific CAR T cells generated with SB and AaPC platforms were safe, and may provide additional cancer control as planned infusions after HSCT. These results support further clinical development of this nonviral gene therapy approach.

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Section: Discussionmentioning

confidence: 99%

Phase I trials using Sleeping Beauty to generate CD19-specific CAR T cells

Kebriaei¹,

Singh

Huls

et al. 2016

Journal of Clinical Investigation

425

390

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“…Conucleofection of both plasmids allowed a 15-fold increase in the number of colonies with stable expression of the transgene when compared to nucleofection of the plasmid of interest alone [17]. Another system, the Sleeping Beauty (SB) transposase/transposon system, has yielded promising results in multipotent adult progenitor cells and human primary T cells [25,26]. In this system, the SB transposase mediates a cut-andpaste mechanism of transposition, which results in the excision of the DNA sequence of interest and its reinsertion into a TA target dinucleotide of the genome.…”

Section: Discussionmentioning

confidence: 99%

Nucleofection Is a Valuable Transfection Method for Transient and Stable Transgene Expression in Adipose Tissue-Derived Stem Cells

Zaragosi¹,

Billon²,

Ailhaud³

et al. 2006

Stem Cells

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“…Since then, it has been used successfully to generate transgenic mice (8) and as a gene therapy vector capable of delivering genes to mouse liver for somatic integration and long-term transgene expression (9). Sleeping Beauty has unique features as a gene therapy vector for a variety of cell targets, including human primary T cells (10). It integrates transgenes randomly into TA-rich regions of the host genome at a frequency comparable with that of viral vectors in both quiescent and replicating cells (9,11).…”

mentioning

confidence: 99%

Erythroid-Specific Expression of β-Globin by the Sleeping Beauty Transposon for Sickle Cell Disease

et al. 2007

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Sickle cell disease (SCD) results predominately from a single monogenic mutation that affects thousands of individuals worldwide. Gene therapy approaches have focused on using viral vectors to transfer wild-type -or γ-globin transgenes into hematopoietic stem cells for long-term expression of the recombinant globins. In this study, we investigated the use of a novel nonviral vector system, the Sleeping Beauty (SB) transposon (Tn) to insert a wild-type -globin expression cassette into the human genome for sustained expression of -globin. We initially constructed a -globin expression vector composed of the hybrid cytomegalovirus (CMV) enhancer chicken -actin promoter (CAGGS) and full-length -globin cDNA, as well as truncated forms lacking either the 3′ or 3′ and 5′ untranslated regions (UTRs), to optimize expression of -globin. -Globin with its 5′ UTR was efficiently expressed from its cDNA in K-562 cells induced with hemin. However, expression was constitutive and not erythroid-specific. We then constructed cis SB-Tn--globin plasmids using a minimal -globin gene driven by hybrid promoter IHK (human ALAS2 intron 8 erythroid-specific enhancer, HS40 core element from human RLCR, ankyrin-1 promoter), IH p (human ALAS2 intron 8 erythroid-specific enhancer, HS40 core element from human RLCR, -globin promoter), or HS3 p (HS3 core element from human LCR, -globin promoter) to establish erythroidspecific expression of -globin. Stable genomic insertion of the minimal gene and expression of the -globin transgene for >5 months at a level comparable to that of the endogenous γ-globin gene were achieved using a SB-Tn -globin cis construct. Interestingly, erythroid-specific expression of -globin driven by IHK was regulated primarily at the translational level, in contrast to post-transcriptional regulation in non-erythroid cells. The SB-Tn system is a promising nonviral vector for efficient genomic insertion conferring stable, persistent erythroid-specific expression of -globin.

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Stable gene transfer and expression in human primary T cells by the Sleeping Beauty transposon system

Cited by 98 publications

References 53 publications

Phase I trials using Sleeping Beauty to generate CD19-specific CAR T cells

Phase I trials using Sleeping Beauty to generate CD19-specific CAR T cells

Nucleofection Is a Valuable Transfection Method for Transient and Stable Transgene Expression in Adipose Tissue-Derived Stem Cells

Erythroid-Specific Expression of β-Globin by the Sleeping Beauty Transposon for Sickle Cell Disease

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