Sleeping Beauty Chi新她meric Antigen Recep多書tor (CAR) Expression Vector

Utilizing chimeric antigen r高商eceptor (CAR) ve友街ctors to produce engineered T cells (金理also known as CAR志師 T cells) that can暗聽 recognize tumor-associated ant空作igens has emerged as a prom視器ising approach in the treatment 花章of cancer. In CAR T-c慢遠ell therapy, T ce南雪lls derived from either 畫妹patients (autologo紅數us) or healthy donors (allogeneic) 答作are modified to express CAR, a子舊 chimeric construct which com相男bines antigen bind通行ing with T cell activation for target舊冷ing tumor cells.

Structurally, a CAR consists of fou算紅r main components: (1) an extracell雜睡ular antigen recognition domain mad離理e up of an antibody-derived single訊日 chain variable fragment (scFv) 數讀of known specif外場icity. The scFv facilitates antigen 會愛binding and is composed of the variab數高le light chain and民離 heavy chain regi問錯ons of an antigen-sp都冷ecific monoclonal antibody co低線nnected by a flexible lin刀問ker; (2) an extracellular h遠錢inge or spacer w西呢hich connects the s拿訊cFv with the transmemb土刀rane domain and provides flexibility a公地nd stability to the CAR structure; 唱報(3) a transmembrane domain which ancho機理rs the CAR to the plasma membrane很水 and bridges the extracellular 離算hinge as well as antigen binding do也農main with the intracellular si冷生gnaling domain. It plays a criti身空cal role in enhancing recepto短又r expression and stabi兵自lity, and (4)an intracell個北ular signaling 微章domain that is typically derived和窗 from the CD3 zeta c那線hain of the T cell receptor (TCR黑時) and contains im山姐munoreceptor tyrosi是視ne-based activation motifs (ITAM家會s). The ITAMs become phospho我森rylated and activate 文友downstream signaling upon antigen bi空湖nding, leading to the subseq數唱uent activation of T ce舊算lls. In addition, the音日 intracellular region may co森遠ntain one or more c自近ostimulatory domains 年票(derived from CD28, CD137,煙看 etc.) in tandem with the CD3 z花友eta signaling domain 男子for improving T cell proliferat區腦ion and persistence.

The structure of CAR ha得男s evolved over the如熱 past few years based on modifica還術tions to the composition of the intrac下唱ellular domains. Th土空e first-generation CARs 物海consisted of on高件ly a single intracellular女又 CD3 zeta-derived signaling domain. W可對hile these CARs 要體could activate T cells, they 話從exhibited poor anti-tumor activi志小ty in vivo due to the low cytot得舞oxicity and proliferation of T cell畫機s expressing such物森 CARs. This led to the advent of路多 second-generation CARs which i音個ncluded an intracellular costimulatory商機 domain in additio船器n to the CD3 zeta signal務哥ing domain leading to a signi技快ficant improveme事們nt in the in vi農務vo proliferation, expansi火是on, and persistence of T ce小月lls expressing second-generation CARs. 司下To further optimize the anti-tum新長or efficacy of 日資CAR-T cells, third-generati近算on CARs were develop林友ed which included two intracellula聽姐r, cis-acting costimulator我鐘y domains in addi長的tion to CD3 zeta. Thereafter, fourth-機遠generation CARs were derived 微錢from second-generation CAR人笑s by modifying their intracell新可ular domain for indu機著cible or constitutive expression 離外of cytokines. The fifth and t嗎化he latest generation哥大 of CARs are also 舊人derived from second-generation C照場ARs by the incor到問poration of intracellul放那ar domains of cytokine receptors.

Our sleeping beauty CAR e業藍xpression vector物影 is a highly efficient tool fo生拿r achieving non-viral, trans藍藍poson-based delivery of second-ge間還neration CAR expression cas妹學settes into T cells. This vecto刀新r system is derived from t劇子he Tc1/mariner superfamily of tra視舞nsposons which were original聽市ly isolated from fish genomes and ha電亮ve been transpositionally inactiv雪醫e due to the accumulation of mutations.你呢 The sleeping bea煙慢uty transposon was reconstruct雜飛ed by eliminating術通 such inactivating mut山紅ations from sequenc也藍es of Tc1/mariner transposons foun車秒d in salmonids.

The sleeping beauty sys地的tem comprises two遠一 components: the transposon plas黑制mid and the transposase (helper). The t明開ransposon plasmid contains 照機two inverted/direct repeats I和近R/DR(R) bracketing the region to be綠相 transposed. The transposa自不se can be delivered i得就nto target cells through two met湖兵hods. A helper plasmid encoding transp習科osase can be transiently 也老transfected into cel章朋ls. Alternatively, target c森月ells can be inje師購cted with in vitro transcribed 的件transposase mRNA. When tran亮東sposon plasmids an友兵d the helper are co-intr見電oduced into target cells, the transpo都小sase produced from the help吧作er would recogni子聽ze the two IR/D友錯R(R)s on the tran生見sposon and insert t書要he flanked region 老金including the two I亮作R/DR(R)s into TA dinucleotide sites of 線煙the host genome. 風用At each insertion site, duplica鐘內ted TA target sites are created, 討下flanking the transpos暗人on in the genome白是. Through both methods of d間購elivering transposase, it is expr上些essed for only a sh場關ort time. Upon the loss of the help問站er plasmid or degrad紅鐵ation of transposas慢購e mRNA, the integration of the trans兵現poson into the host事在 genome becomes permanent生站.

Sleeping Beauty is a class II transpos視和on, meaning that可討 it moves in a cut-and-paste man得舞ner, hopping from pl房筆ace to place without leaving copies b動是ehind (In contrast, class I transpo工森sons move in a copy-and-pas離船te manner). If t農舊he sleeping beauty t子醫ransposase is reintrod說火uced into the cells, t子用he transposon could be excised fr做熱om the genome of鄉年 some cells. The excision results in 子如the formation of a “transposon footpri司場nt”, consisting of three nucleotides 紙時flanked by duplicated TA target sit妹討es.

For further information about this v拿們ector system, please refer to the pap道又ers below.

Permanent integrati的家on of vector DNA: The sleeping beau工亮ty CAR expression vector helps achiev我視e long-term expression of CAR exp這我ression cassettes in T cells by al那哥lowing permanent integration of the t公腦ransposon carrying the CAR cassette int嗎中o the host cell genome.

Technical simpl鐵雜icity: Sleeping beauty-based CAR vect信秒ors can be deliver鐵還ed into T cells by electrop農購oration which is technically 很熱simpler compared to using viru現體s-based CAR constructs. Viral vecto南些rs require the packaging of liv女草e virus before t們相hey can be transduced into 銀資T cells, making th一頻e process technically challengi車我ng and time-consuming.

Low cost: Manufacturing of clinical-黑相grade sleeping beauty vect她還ors is far less expens湖西ive than viral vect市東ors which makes clinical developme舞現nt of piggyBac-based CAR T cells much 房金more cost-effective. This offe知放rs a significant advantage in t算件he development of C很師AR T therapies since th樹刀e clinical efficacy of CAR T cells學懂 cannot be accurately predic冷月ted by preclinical stu河術dies in mice models and should b靜數e determined by clinic中銀al studies.

Delivery method: Sleeping beauty-based CAR expres腦對sions vectors are typi可湖cally delivered into T cells by elec著區troporation which typically 鐵裡results in a low to medi暗上um frequency of cells w上些ith successful 文匠integration of the間遠 CAR expression casset吃日te. Additionally, electropo什通ration can be toxic to T cel刀腦ls which in turn could lead森動 to reduced yiel男輛d of functional CA姐妹R T cells. Therefore, it may be和朋 necessary to culture the T cells 他金ex vivo for extended periods to attain草近 the quantities needed for infusion, w微爸hich over time could哥費 alter cell phenotype leading to 吧家reduced long-term memo少技ry function.

Limited transposon carrying capacity: For transposons be遠但tween 1.9 and 7.2 Kb, transpo慢玩sition frequency decreases with incre就弟ase in transposon length.