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.

Prommersberger S ，Monjezi R ，Shankar R ，Schmeer M ，Hudecek M ，Ivics Z ，Schleef M ... -  《-》

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

Hudecek M ，Gogishvili T ，Monjezi R ，Wegner J ，Shankar R ，Kruesemann C ，Miskey C ，Ivics Z ，Schmeer M ，Schleef M ... -  《-》

Engineering of potent CAR NK cells using non-viral Sleeping Beauty transposition from minimalistic DNA vectors.

Natural killer (NK) cells have high intrinsic cytotoxic capacity, and clinical trials have demonstrated their safety and efficacy for adoptive cancer therapy. Expression of chimeric antigen receptors (CARs) enhances NK cell target specificity, with these cells applicable as off-the-shelf products generated from allogeneic donors. Here, we present for the first time an innovative approach for CAR NK cell engineering employing a non-viral Sleeping Beauty (SB) transposon/transposase-based system and minimized DNA vectors termed minicircles. SB-modified peripheral blood-derived primary NK cells displayed high and stable CAR expression and more frequent vector integration into genomic safe harbors than lentiviral vectors. Importantly, SB-generated CAR NK cells demonstrated enhanced cytotoxicity compared with non-transfected NK cells. A strong antileukemic potential was confirmed using established acute lymphocytic leukemia cells and patient-derived primary acute B cell leukemia and lymphoma samples as targets in vitro and in vivo in a xenograft leukemia mouse model. Our data suggest that the SB-transposon system is an efficient, safe, and cost-effective approach to non-viral engineering of highly functional CAR NK cells, which may be suitable for cancer immunotherapy of leukemia as well as many other malignancies.

Bexte T ，Botezatu L ，Miskey C ，Gierschek F ，Moter A ，Wendel P ，Reindl LM ，Campe J ，Villena-Ossa JF ，Gebel V ，Stein K ，Cathomen T ，Cremer A ，Wels WS ，Hudecek M ，Ivics Z ，Ullrich E ... -  《-》

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

Monjezi R ，Miskey C ，Gogishvili T ，Schleef M ，Schmeer M ，Einsele H ，Ivics Z ，Hudecek M ... -  《-》

Generation of CAR-T Cells with Sleeping Beauty Transposon Gene Transfer.

Human T lymphocytes that transgenically express a chimeric antigen receptor (CAR) have proven efficacy and safety in gene- and cell-based immunotherapy of certain hematological cancers. Appropriate gene vectors and methods of genetic engineering are required for therapeutic cell products to be biologically potent and their manufacturing to be economically viable. Transposon-based gene transfer satisfies these needs, and is currently being evaluated in clinical trials. In this protocol we describe the basic Sleeping Beauty (SB) transposon vector components required for stable gene integration in human cells, with special emphasis on minicircle DNA vectors and the use of synthetic mRNA. We provide a protocol for functional validation of the vector components in cultured human cell lines on the basis of fluorescent reporter gene expression. Finally, we provide a protocol for CAR-T cell engineering and describe assays that address transgene expression, biological potency and genomic vector copy numbers in polyclonal cell populations. Because transposons allow virus-free gene transfer with naked nucleic acids, the protocol can be adopted by any laboratory equipped with biological safety level S1 facilities.

Prommersberger S ，Monjezi R ，Botezatu L ，Miskey C ，Amberger M ，Mestermann K ，Hudecek M ，Ivics Z ... -  《-》