Evolution of the clinical-stage hyperactive TcBuster transposase as a platform for robust non-viral production of adoptive cellular therapies.

Cellular therapies for the treatment of human diseases, such as chimeric antigen receptor (CAR) T and natural killer (NK) cells have shown remarkable clinical efficacy in treating hematological malignancies; however, current methods mainly utilize viral vectors that are limited by their cargo size capacities, high cost, and long timelines for production of clinical reagent. Delivery of genetic cargo via DNA transposon engineering is a more timely and cost-effective approach, yet has been held back by less efficient integration rates. Here, we report the development of a novel hyperactive TcBuster (TcB-M) transposase engineered through structure-guided and in vitro evolution approaches that achieves high-efficiency integration of large, multicistronic CAR-expression cassettes in primary human cells. Our proof-of-principle TcB-M engineering of CAR-NK and CAR-T cells shows low integrated vector copy number, a safe insertion site profile, robust in vitro function, and improves survival in a Burkitt lymphoma xenograft model in vivo. Overall, TcB-M is a versatile, safe, efficient and open-source option for the rapid manufacture and preclinical testing of primary human immune cell therapies through delivery of multicistronic large cargo via transposition.

Skeate JG ，Pomeroy EJ ，Slipek NJ ，Jones BJ ，Wick BJ ，Chang JW ，Lahr WS ，Stelljes EM ，Patrinostro X ，Barnes B ，Zarecki T ，Krueger JB ，Bridge JE ，Robbins GM ，McCormick MD ，Leerar JR ，Wenzel KT ，Hornberger KM ，Walker K ，Smedley D ，Largaespada DA ，Otto N ，Webber BR ，Moriarity BS ... -  《-》

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 ... -  《-》

CAR T Cells Generated Using Sleeping Beauty Transposon Vectors and Expanded with an EBV-Transformed Lymphoblastoid Cell Line Display Antitumor Activity In Vitro and In Vivo.

Chicaybam L ，Abdo L ，Carneiro M ，Peixoto B ，Viegas M ，de Sousa P ，Fornazin MC ，Spago MC ，Albertoni Laranjeira AB ，de Campos-Lima PO ，Nowill A ，Barros LRC ，Bonamino MH ... -  《-》

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 ... -  《-》

Immunotherapy of acute leukemia by chimeric antigen receptor-modified lymphocytes using an improved Sleeping Beauty transposon platform.

Magnani CF ，Turazzi N ，Benedicenti F ，Calabria A ，Tenderini E ，Tettamanti S ，Giordano Attianese GM ，Cooper LJ ，Aiuti A ，Montini E ，Biondi A ，Biagi E ... -  《Oncotarget》