PiggyBac Chimeric Antige件報n Receptor (CAR) Expression 小她Vector

Utilizing chimeri麗他c antigen receptor (CAR) ve店多ctors to produce engineered T cells (事睡also known as CAR T cells) that 科在can recognize tumor-as匠是sociated antigens has emerged as a老筆 promising appr男頻oach in the trea一用tment of cancer. In CAR T-cell 醫友therapy, T cells derived from either作吧 patients (autologous) or h街厭ealthy donors (a綠子llogeneic) are modified to expr外市ess CAR, a chime還腦ric construct which combines antige多內n binding with T cell activ吃很ation for targeting tu新廠mor cells.

Structurally, a CAR co拍我nsists of four 請笑main components: (1) an extracellula很紅r antigen recog是術nition domain made u不從p of an antibody-deri用水ved single chain 睡可variable fragment (scFv) of known spec河廠ificity. The scFv fac他舊ilitates antigen b小身inding and is composed of the兒鐘 variable light chain生話 and heavy chain regions of an anti妹老gen-specific monoclonal ant森劇ibody connected by a fl和一exible linker; (2) an extr開秒acellular hinge or spacer whi機科ch connects the scFv with the transmemb照劇rane domain and provides flexi數花bility and stability t關銀o the CAR structure; (3) a t和美ransmembrane domain whi女們ch anchors the CAR to 車樂the plasma membrane 器了and bridges the extracellular hin就窗ge as well as antigen binding domain wi快愛th the intracellular signaling doma工技in. It plays a cri路秒tical role in enhancing receptor e理關xpression and stability, 近制and (4) an intracellular signaling doma門國in that is typically deriv船匠ed from the CD3 z愛聽eta chain of the T cell recept雪技or (TCR) and contains immunoreceptor t師問yrosine-based a為錢ctivation motifs (ITAMs). The IT就很AMs become phosphorylated and act能吧ivate downstream signaling錢些 upon antigen b了地inding, leading to the subsequent大討 activation of T cells. In a物答ddition, the intracellular region m微黃ay contain one or mor的快e costimulatory domains (derived小科 from CD28, CD137, etc.) in tandem with小新 the CD3 zeta signali很算ng domain for improving會作 T cell proliferation 玩著and persistence.

The structure o木能f CAR has evolved人如 over the past few years based on mo文少difications to the compo一書sition of the intrac愛很ellular domains. The firs海音t-generation CA玩師Rs consisted of only a sin西坐gle intracellular CD3 zeta-derive東裡d signaling domain. While線鐵 these CARs could act不懂ivate T cells, t物校hey exhibited poor anti-tumor acti高笑vity in vivo due to th數時e low cytotoxicity and prolife兒紙ration of T cells expressing such CAR們司s. This led to the advent of s文為econd-generation CA為個Rs which included an intr樂土acellular costimulatory do水林main in addition to the CD3 zeta si農紙gnaling domain le醫行ading to a significant impr煙光ovement of the in vivo proli近草feration, expansion, and persistence o黃城f T cells expre房如ssing second-generation CARs. To furth讀術er optimize the anti-服廠tumor efficacy 著話of CAR-T cells, 近近third-generation CARs were developed w數能hich included two intracellular腦弟, cis-acting costimula門火tory domains in addition to CD3 zeta請市. Thereafter, fourth-generat雜可ion CARs were derived 問輛from second-generation CARs by modif如光ying their intracellular domain for 明腦inducible or constit裡匠utive expression 長在of cytokines. The fifth an去秒d the latest gene快司ration of CARs are also der跳下ived from second計她-generation CARs by the incorpor雜去ation of intracellular domains of cytok話花ine receptors.

Our piggyBac CAR expression vecto風也r is a highly effici我地ent tool for ach兒吧ieving non-viral, transpo大線son-based delivery o女可f second-generation CAR e坐都xpression cassettes into 內體T cells. The piggyBac system is der不術ived from the piggyBac transposon,花農 which is originall區照y isolated from the 電車cabbage looper (Trichoplusia ni; 術土a moth species).

The piggyBac system玩務 comprises two componen南木ts: the transposon plasmid and the t算懂ransposase (helper PBase).都員 The transposon plasmid contain人醫s two terminal repeat我紙s (TRs) bracketing the region to be t短分ransposed. The transposase妹區 can be delivered into target cells t日窗hrough two methods.志玩 A helper plasmid encoding 影術PBase can be tran飛離siently transfected村費 into cells. Alternati雨鐘vely, target cells can be injected wit為空h in vitro trans好金cribed PBase mRNA老會. When the helper PBase and the 區器piggyBac transposon vector 喝喝are co-introduced into target cells見雜, the transposase produced fr件外om the helper would recognize北黑 the two TRs on the transposon an離藍d insert the flanked region 雜水including the two TR快和s into the host genome. Insertion 妹玩typically occurs at host chromosoma飛街l sites that contain 雨拍the TTAA sequence, which is d工火uplicated on the two flanks of the in冷章tegrated fragment. Through both met身我hods of delivering transposase, i河器t is expressed for only a short t樹聽ime. Upon the loss of the helper pla司行smid or degradat路民ion of transposase mRNA,年放 the integration of the transpos黑下on into the host genome be做懂comes permanent.

PiggyBac is a class II理房 transposon, me笑志aning that it moves in a cut-and-pas愛美te manner, hopping from place to話在 place without leaving copi電他es behind. (In 些吧contrast, class I transposons move in 木南a copy-and-paste man路城ner.) If the transp司弟osase is reintroduce都都d into the cells, the tr什山ansposon could get exc器店ised from the genome of some cells, fo說遠otprint-free.

For further information ab行門out this vector system, please refe分體r to the papers below.