A highly soluble Sleeping Beauty transposase improves control of gene insertion

Querques, Irma; Mades, Andreas; Zuliani, Cecilia; Miskey, Csaba; Alb, Miriam; Grueso, Esther; Machwirth, Markus; Rausch, Tobias; Einsele, Hermann; Ivics, Zoltán; Hudecek, Michael; Barábas, O.

doi:10.1038/s41587-019-0291-z

Cited by 77 publications

(63 citation statements)

References 77 publications

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“…The use of transposon containing DNA minicircles and transposase proteins has shown to improve the cell viability of electroporated T cells. 17,53 Furthermore, providing antigen-presenting cells (APCs) in vitro to freshly electroporated T cells can help preserving cell viability. 16 Since in our POC approach T cells will likely encounter APCs in vivo after infusion, this could further increase gene-modified cell function, viability, and persistence.…”

Section: Comparison Of Anti-tumor Activity Of Cells 4 H Vs or 24 H Pomentioning

confidence: 99%

Development of CAR-T cell therapy for B-ALL using a point-of-care approach

et al. 2020

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Recently approved by the FDA and European Medicines Agency, CART cell therapy is a new treatment option for B-cell malignancies. Currently, CART cells are manufactured in centralized facilities and face bottlenecks like complex scaling up, high costs, and logistic operations. These difficulties are mainly related to the use of viral vectors and the requirement to expand CART cells to reach the therapeutic dose. In this paper, by using Sleeping Beauty-mediated genetic modification delivered by electroporation, we show that CART cells can be generated and used without the need for ex vivo activation and expansion, consistent with a point-of-care (POC) approach. Our results show that minimally manipulated CART cells are effective in vivo against RS4;11 leukemia cells engrafted in NSG mice even when inoculated after only 4 h of gene transfer. In an effort to better characterize the infused CART cells, we show that 19BBz T lymphocytes infused after 24 h of electroporation (where CAR expression is already detectable) can improve the overall survival and reduce tumor burden in organs of mice engrafted with RS4;11 or Nalm-6 B cell leukemia. A side-by-side comparison of POC approach with a conventional 8-day expansion protocol using Transact beads demonstrated that both approaches have equivalent antitumor activity in vivo. Our data suggest that POC approach is a viable alternative for the generation and use of CART cells, overcoming the limitations of current manufacturing protocols. Its use has the potential to expand CAR immunotherapy to a higher number of patients, especially in the context of low-income countries.

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Section: Comparison Of Anti-tumor Activity Of Cells 4 H Vs or 24 H Pomentioning

confidence: 99%

Development of CAR-T cell therapy for B-ALL using a point-of-care approach

et al. 2020

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“…In the corresponding article, the researchers used the transcriptional activator M2/hyperactive Sleeping Beauty/ transposon (M2/HSB/Tn) mouse model, random transposon integration was induced by adding doxycycline, and red fluorescent protein was used to label cells to track blood progenitor cells during blood regeneration [13,47]. Subsequently, a new type of Sleeping Beauty transposon was developed to generate chimeric antigen receptor T cells and applied in cancer immunotherapy [48]. Previously, due to the lack of appropriate tools to study the natural formation of blood cells, possibly highlighting the practicality and innovation of this technology, significant advances have been made in both therapeutics and gene editing, as it improves fidelity and increases the safety of genomic modifications.…”

Section: Barcode Technologymentioning

confidence: 99%

Lineage tracing: technology tool for exploring the development, regeneration, and disease of the digestive system

et al. 2020

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Lineage tracing is the most widely used technique to track the migration, proliferation, and differentiation of specific cells in vivo. The currently available gene-targeting technologies have been developing for decades to study organogenesis, tissue injury repairing, and tumor progression by tracing the fates of individual cells. Recently, lineage tracing has expanded the platforms available for disease model establishment, drug screening, cell plasticity research, and personalized medicine development in a molecular and cellular biology perspective. Lineage tracing provides new views for exploring digestive organ development and regeneration and techniques for digestive disease causes and progression. This review focuses on the lineage tracing technology and its application in digestive diseases.

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“…SB11 transposase-expressing plasmid, though bearing the transiently active cytomegalovirus early promoter, causes at low frequency extended transposase protein expression which could potentially lead to remobilization of the transposon in other genomic compartments(34). For a further platform implementation, the hyperactive SB100X(35) variant in mRNA(36) or protein(37) version could be used. These alternatives may lead to a level of integration of SB minicircles up to 45% and 20-30%, respectively, thus avoiding the theoretical risk of chromosomal integration, and reducing the time of in vitro culturing.The rationale of using donor-derived memory T cells as CIK cells over conventional CAR T cells comes from clinical experiences clearly demonstrating safety and tolerability of repeated CIK cell infusions with minimal and manageable GVHD occurrence(17,18,38).…”

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confidence: 99%

Sleeping Beauty–engineered CAR T cells achieve antileukemic activity without severe toxicities

Magnani¹,

Gaipa²,

Lussana³

et al. 2020

Journal of Clinical Investigation

134

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Background. Chimeric antigen receptor (CAR) T cell immunotherapy has achieved complete remission and durable response in highly refractory patients. However, logistical complexity and high costs of manufacturing autologous viral products limit CAR T cell availability. Methods. We reported the early results of a phase I/II trial in B-cell acute lymphoblastic leukemia (B-ALL) patients relapsed after allogeneic hematopoietic stem cell transplantation (HSCT) using donor-derived CD19 CAR T cells generated with the Sleeping Beauty (SB) transposon and differentiated into cytokine induced killer cells (CIK). Results. The cellular product was produced successfully for all patients from the donor peripheral blood (PB) and consisted mostly of CD3+ lymphocytes with 43% CAR expression. Four pediatric and 9 adult patients were infused with a single dose of CAR T cells. Toxicities reported were two grade I and a grade II cytokine release syndrome (CRS) cases at the highest dose, in the absence of graftversus-host disease (GvHD), neurotoxicity, or dose-limiting toxicities. Six out of 7 patients, receiving the highest doses, achieved CR and CRi at day 28. Five out of 6 patients in CR were also minimal residual disease (MRD)-negative. Robust expansion was achieved in the majority of the patients. CAR T cells were measurable by transgene copy PCR up to 10 months. Integration site analysis showed a positive safety profile and highly polyclonal repertoire in vitro and at early time points after infusion. Conclusion. SB-engineered CAR T cells expand and persist in pediatric and adult BALL patients relapsed after HSCT. Anti-leukemic activity was achieved without severe toxicities. Trial registration. clinicaltrials.gov NCT03389035.

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A highly soluble Sleeping Beauty transposase improves control of gene insertion

Cited by 77 publications

References 77 publications

Development of CAR-T cell therapy for B-ALL using a point-of-care approach

Development of CAR-T cell therapy for B-ALL using a point-of-care approach

Lineage tracing: technology tool for exploring the development, regeneration, and disease of the digestive system

Sleeping Beauty–engineered CAR T cells achieve antileukemic activity without severe toxicities

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