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哺乳動物CAR基因表達自失活型MMLV載體
概述
Utilizing chimeric antigen 去玩receptor (CAR) vectors to produce engi電爸neered T cells (also known as CAR T水那 cells) that can recognize tumo家下r-associated antigens h玩子as emerged as a p關鄉romising approach in the treat花音ment of cancer. In都多 CAR T-cell the為上rapy, T cells deri影是ved from either patients (autologous) 也很or healthy donors (allogeneic)國南 are modified to express 個短CAR, a chimeric construct which志明 combines antig線熱en binding with T cell activation下機 for targeting t匠現umor cells.
Structurally, a CAR consists of four問個 main components: (1) an理頻 extracellular antigen recognition do快快main made up of an antibody-der鄉也ived single chain variable fragm美那ent (scFv) of known specificity.明慢 The scFv facilitates antigen b吧師inding and is composed of the variable家看 light chain and 就去heavy chain regions o志會f an antigen-spe女醫cific monoclonal antibody connected b外內y a flexible linker; (2) an extracel森錯lular hinge or spacer which connects th店務e scFv with the 什體transmembrane domain and provides fl木地exibility and stabili木新ty to the CAR structure; (3)白西 a transmembrane domain which a城離nchors the CAR to t微吃he plasma membr街麗ane and bridges the extracellular hin樂海ge as well as a飛為ntigen binding 店友domain with the intracellular 會銀signaling domain. 電討It plays a critical rol下醫e in enhancing receptor expr坐媽ession and stability; (4) a路內nd an intracellular signaling domain 西數which is typically derived from the C火醫D3 zeta chain of the T cell receptor (T不鄉CR) and contains immunoreceptor訊歌 tyrosine-based activation m電計otifs (ITAMs). The ITAMs becom務舊e phosphorylated大聽 and activate dow嗎站nstream signaling upo對紙n antigen binding, leading to the s腦金ubsequent activ山志ation of T cells. In 友樂addition, the intracellular region may銀行 contain one or more costimulatory doma近河ins (derived from CD28, CD137 etc.) i你話n tandem with the CD3 zeta 個事signaling domain for impro答關ving T cell proliferation and persist頻靜ence.
The structure of CAR has evolved o火文ver the past few years based o懂遠n modifications to 呢輛the composition of the intracel關好lular domains. The fi還雨rst-generation CARs consisted of影你 only a single in微綠tracellular CD3 zet章生a-derived signaling domain. While 知機these CARs could activa裡地te T cells, they e廠森xhibited poor ant司到i-tumor activity in vivo due to那司 the low cytotoxicity and proliferati你為on of T cells expressin房吃g such CARs. This led to the adven門藍t of the second-generat現金ion CARs which included a術什n intracellular costimulatory d藍看omain in addition to the CD3 zeta sig東風naling domain leading to 房但a significant improvement in th們費e in vivo proliferation行票, expansion and pers子開istence of T cells現高 expressing second gen短我eration CARs. To further 愛美optimize the anti愛市-tumor efficacy of C計可AR-T cells, thi朋花rd generation CARs we歌就re developed which included腦靜 two intracellular, cis技她-acting costimulat去視ory domains in add厭笑ition to CD3 ze鐵音ta. Thereafter, fourth genera事國tion CARs were derived f拍店rom second-generation他兒 CARs by modifying their int票多racellular domain f水紅or inducible or constitutive expr從物ession of cytokines. The fifth and the 舊黑latest generation 吃煙of CARs are also derived from second-g算冷eneration CARs by t城讀he incorporation of相森 intracellular domains of c票員ytokine receptors.
Our MMLV retrovirus CAR expression ve術購ctor is derived from行飛 the Moloney murine leukemia virus,務能 which is a member of the retrovirus f員劇amily and is highly suita西草ble for retrovi金山rus-mediated delivery of 區來second-generation CAR exp業聽ression cassettes生白 into T cells.
MMLV, a retroviral ve器畫ctor derived from Moloney murine leuk劇關emia virus, is 到現a plus-strand linear RNA 輛書virus that exhibit村內s efficient genomic inte男綠gration. While our wildtype科相 MMLV retrovirus ex好雨pression vector間事 utilizes the ubiquitous p小南romoter function i風多n the 5' long terminal re愛中peat (LTR) of wildty鄉做pe MMLV genome for driving expr鄉長ession of the CAR cassette, the sel我對f-inactivating MM討計LV retrovirus expression vector allows國高 users to select any promote店師r of their choice f站機or driving CAR expression. Thi店廠s is achieved by the dele媽火tion of the U3 region in the MMLV玩還 3’ LTR which self-inactiva火從tes the promoter activity訊通 in the 5' LTR by a copying 都日mechanism during v書拿iral genome integrati城什on. This not only provides user門票s with the flexibility to add th冷城eir promoter of choice for文從 driving CAR expression空是 but also elimin我件ates the risk of oncogen遠白ic activation of理空 adjacent genes upon vector integr秒藍ation, thereby enabling suc坐子h vectors to have a higher safety profi中黑le compared to wild視從type MMLV vectors.
The self-inactivating MMLV retrov子問irus CAR expres機放sion vector is f器愛irst constructed 林船as a plasmid in E. coli whe藍和re the entire CAR expression casse文們tte including the s生關cFv region, hinge, transmembrane doma坐讀in and intracellula裡村r CD3 zeta signaling domain習笑 as well as the co文請stimulatory domain is clon明近ed in between the知體 two MMLV LTRs. It is們行 then transfected into packaging c區紙ells along with several helpe明去r plasmids. Inside t微內he packaging cells, vector DNA 學林located between the two LTRs is tra對自nscribed into RNA, and vi店用ral proteins expressed by the helper 路鄉plasmids further package the RNA in城體to virus. Live virus is the制員n released into t姐相he supernatant, whic從動h can be used to infect targe也了t cells directly or after concentr老長ation. When the virus is ad土不ded to target cells, the RNA car腦月go is shuttled into cells where it i姐見s reverse transcribed into DNA畫些 and randomly integ時筆rated in the host genome. Any gene(s) t草腦hat were placed in-between the tw在能o LTRs during vector cl音可oning are permanently朋我 inserted into host DNA alongsid下火e the rest of viral genom可玩e.
By design, self-inactiva外我ting MMLV retrovi女鄉ral vectors lack the genes req上請uired for viral packaging 車自and transduction輛地 (these genes are carried by h些又elper plasmids o們藍r integrated into p厭視ackaging cells instead). A中短s a result, viruses錢報 produced from thes員從e vectors have the兵會 important safety feature of bei電資ng replication incompetent (票月meaning that they can transduce targe匠見t cells but cannot re日機plicate in them).
For further information about木水 this vector system, please r好朋efer to the papers b銀到elow.
| References | Topic |
|---|---|
| Br J Cancer. 120:26 (2019)廠朋 | Review on next-genera動員tion CAR T cells |
| Mol Ther Oncolytics. 3:16014 謝答(2016) | Review on CAR mo明在dels |
| J Immunother. 32:169 男技(2009) | MMLV retrovirus-mediated CAR e要視xpression for autologous adoptive ce內弟ll therapy |
| J Immunother. 32:68機喝9 (2009) | Construction and pre-clinical evalu西快ation of an anti-CD報醫19 CAR |
| Mol Ther. 11:1919 (2009)飛雪 | Insertional transformation 雨舊of HSCs by SIN MMLV |
亮點
Our SIN MMLV retrovir長間us CAR expression vector can be used f唱就or the expression of拍哥 second-generation C用用ARs. It is optimized for high copy numb山水er replication in E. coli, high-titer下都 packaging of live virus, effi遠員cient viral transduction o數又f a wide range of cells,房雨 efficient vector integration into t鐘醫he host genome, 花訊and high-level transge可農ne expression.
優勢
Permanent integration of vector DN車空A: Conventional transfection res廠店ults in almost 呢愛entirely transient delivery我土 of DNA into host cel資老ls due to the los歌黑s of DNA over time. This prob林音lem is especially prominent 是區in rapidly dividing cells. In c學森ontrast, retroviral transduction can d計個eliver genes permanently into host c去能ells due to inte見呢gration of the viral v街身ector into the host 時是genome, thereby e什個nabling long-term expression of CAR 風火expression cassettes.
Broad tropism: 兒很; Our packaging間人 system adds the VSV-事厭G envelop protein to the viral sur你章face. This protein has broad tropi線看sm. As a result, cells from all com風校monly used mammalian sp用公ecies such as huma視術n, mouse and rat ca工錢n be transduced. Furthermore, many spec舊風ific cell types can be t畫身ransduced, though our vecto廠業r has difficulty transducin外身g non-dividing cells 下不(see disadvantages below).
Customizable internal promote關跳r: Our vector is designed to self-海草inactivate the p快樹romoter activity in its 5'區火 LTR upon integration in老坐to the genome. As a result, users can報聽 put in their own promoter to dr視區ive their CAR expression cassette 火他within the vector. Th腦術is is a distinct advantage o飛雨ver our wildtype MMLV retrovirus vect費劇ors, which rely on the promoter f理刀unction of 5' LTR to綠下 drive the ubiquitous expression海國.
Relative uniformity of delivery: 照微; Generally, viral transduction c微男an deliver vectors int吃我o cells in a relat行坐ively uniform manner. In cont我劇rast, conventional tr看們ansfection of plasmid vectors can 務亮be highly non-uniform, with so照可me cells receiv有件ing a lot of copies while照中 other cells receiving few co件上pies or none.
Effectiveness in vitr愛化o and in vivo: While our vecto水放r is mostly used for in vitro transduct亮呢ion of cultured cells, it can亮她 also be used to transduce cells in liv錯場e animals.
Safety: The safety of our vector is ensur一事ed by two features. One is小厭 the partitioning of genes required 煙房for viral packaging and transduction弟線 into several helper plasmid民看s; the other is self-inactivation o做就f the promoter activity in t信我he 5' LTR upon vector inte熱來gration. As a re了資sult, it is essentially im風機possible for replication co用北mpetent virus to emerge 會我during packaging and tran高高sduction. The health risk of麗用 working with our vector is筆遠 therefore minimal.
不足之處
Moderate viral t車呢iter: Viral titer from商地 our vector reaches ~107 TU/ml in the supernatant of海森 packaging cells without furth音熱er concentration. This 醫數is about an order of magnitude lo舞些wer than our lentiviral vectors.&nb生日sp;
More limited cargo space than wildt做可ype MMLV: The MMLV retroviral genome is ~8朋民.3 kb. In our vector, the compon匠場ents necessary for viral pa睡化ckaging and transduc紅員tion occupy ~2.6-3 kb, w些妹hich leaves only ~5.3-5.7 kb to森謝 accommodate the user's D厭還NA of interest, includin靜樹g both the CAR ex河微pression cassette and the pro站關moter.
Difficulty transducing n外動on-dividing cells: Our vector has diffic我火ulty transducing non-dividing cells.&n信到bsp;
Technical complexity: The use of 中也MMLV retroviral vectors requires th開讀e production of live virus in pac費姐kaging cells followed by the 農身measurement of viral titer. T分信hese procedures are technical窗計ly demanding and time consuming relati問車ve to conventional plasmid transfe數房ction.
載體關鍵元件
CMV promoter: Human cytomegalovirus imm話物ediate early promoter為很. It drives transcription of viral RNA媽討 in packaging cells. This RNA is then p從靜ackaged into live virus.
MMLV 5' LTR-ΔU3: A deleted version of the MMLV re高房trovirus 5' long terminal repeat. I能筆n wildtype MMLV retrovirus長雨, 5' LTR and 3'男車 LTR are essentially identical in 呢算sequence. They reside on two end睡通s of the viral genome and point in the 老睡same direction. U去算pon viral integrati金就on, the 3' LTR sequen的就ce is copied on紙習to the 5' LTR. The LTRs carry both pro吧爸moter and polyadenylation f作這unction, such that the 5' LTR a工事cts as a promoter to drive the線光 transcription of the 場能viral genome, while 藍舊the 3' LTR acts as a polyadenyl姐醫ation signal to動身 terminate the upstream tra科放nscript. On our vector,相謝 MMLV 5' LTR-ΔU3 is delete子朋d for a region that is required f懂那or the LTR's promoter a些司ctivity. This does n說費ot affect the production of viral RNA d拿鄉uring packaging because the 地黃promoter function i中通s supplemented by the CMV promoter engi市山neered upstream o錯醫f Δ5' LTR.
Ψ plus pac商務k2: MMLV retrovirus packagi行業ng signal required 說老for the packaging報答 of viral RNA into virus.說區
Promoter: The promoter driv志事ing your gene of in不河terest is placed h行學ere.
Kozak: Kozak consensus sequence.著就 It is placed in front of the start co時畫don of the ORF of i窗吧nterest because it is believed to facil物哥itate translation initi自西ation in eukaryotes.
CD8-leader: Leader signal pepti音弟de of T-cell surface glycoprote吃照in CD8 alpha chain. Directs transp弟工ort and localization of th制街e protein to the 煙日T-cell surface.
scFv: Single chain variable 玩不fragment derived f可小rom a monoclonal ant山頻ibody of known specific遠我ity. Recognizes cells in an antigen-spe煙河cific manner.
Hinge: Extracellular hinge region of the紙中 CAR. Connects scFv with the trans紅爸membrane region providing stability 站了and flexibility for e厭玩fficient CAR expression and function多兒; enhances efficiency of tum機南or recognition; improves expansion of C睡農AR-T cells.
Transmembrane domain: Transmembrane domain of the CAR通拿. Anchors the CAR to the plasma 間看membrane and bridges the extracell聽討ular hinge as well a快學s antigen recognition do物低mains with the intracellular signal土員ing region; enha鐘吃nces receptor ex音裡pression and stability.
Costimulatory domain:湖他 Intracellular costimulatory 音著domain of the CAR. Improv銀身es overall survival, prolif西外eration, and persis拍風tence of activated CAR-T cells.
CD3zeta: Intracellu為匠lar domain of the T cell r體飛eceptor-CD3ζ chain. Acts as a s計西timulatory molecule for 嗎爸activating T cell-mediated im煙窗mune response.
WPRE: Woodchuck hepatitis virus posttranscr內可iptional regulatory element.雜林 It enhances viral RNA stability 舞懂in packaging cells, lea離做ding to higher titer of packaged viru開西s.
MMLV 3' LTR-ΔU3:&nbs照算p;A truncated version of the MMLV re請那trovirus 3' long terminal repeat. This 員刀leads to the self-inactivation 的議of the promoter activity of the 5'市她 LTR upon viral 個林vector integration into生數 the host genome (due to the fact that 畫答3' LTR is copied ont頻女o 5' LTR during v計數iral integration). The polyadenyl算志ation signal contained i員可n MMLV 3' LTR-ΔU3 serves可能 to terminates all upstream transcripts科錢 produced both duri得低ng viral packagin照做g and after viral int城影egration into th刀冷e host genome.
SV40 late pA:&nbs來城p;Simian virus 40 late polyadenylation si志到gnal. It further facilita麗答tes transcriptional terminati遠去on after the 3' LTR d機話uring packaging. Th雜視is elevates the level of function刀服al viral RNA in packaging cells, th中用us improving viral titer女兒.
pUC ori: pUC origin of replication. Plasm人年ids carrying this origin ex自作ist in high copy numbers 志腦in E. coli.
Ampicillin: Ampicillin resi科北stance gene. It allows子近 the plasmid to be maintained by 了物ampicillin selection in E. coli.
Mammalian Chimeric Antigen Receptor (CA綠兒R) Expression Self-Inactivating MMLV Ve聽工ctor
概述
Utilizing chimeric antigen rec樂章eptor (CAR) vectors to produce 事習engineered T cells劇窗 (also known as CAR T家聽 cells) that can r藍熱ecognize tumor-低新associated antigens has emerged as a很遠 promising approach in the treatm花舞ent of cancer. In CAR T-cel玩火l therapy, T cells derived from eith就民er patients (autologous) or healt西們hy donors (allogeneic) are mo弟是dified to express CAR, a chimer時微ic construct which combines antig要山en binding with T cell activatio腦大n for targeting tumor cells.
Structurally, a CAR consists of fou山醫r main components: (1) an extracel有又lular antigen recognition domain made 信生up of an antibody-derived single chai弟兒n variable fragmen月慢t (scFv) of known spec暗去ificity. The scFv facilitate朋光s antigen binding and is com很長posed of the variable li秒村ght chain and hea離會vy chain region物數s of an antigen-specific mono拍討clonal antibody connected by a fl對如exible linker; (2) an extracel體書lular hinge or space爸呢r which connects the scFv with the 線信transmembrane domain懂森 and provides flexibil草農ity and stability t外件o the CAR struct請樂ure; (3) a transmem短藍brane domain which anchors the 電舊CAR to the plasma membrane 了舊and bridges the extr麗訊acellular hinge刀厭 as well as antigen bindi樹和ng domain with the intracellu綠討lar signaling domain. It plays a 低湖critical role in enhan化外cing receptor expression and stabili生新ty; (4) and an intracellular s件人ignaling domain which is typically 票女derived from the CD3 zeta chain o嗎音f the T cell receptor (TCR) and cont數劇ains immunoreceptor tyrosine吧跳-based activation mot鐵照ifs (ITAMs). The ITAMs購吃 become phosphorylated and activate do呢的wnstream signaling upo票兵n antigen binding, leading to the su身厭bsequent activation of T開線 cells. In addition, the i藍刀ntracellular region may contain o小女ne or more costimulatory domains間高 (derived from CD28, CD137 etc好現.) in tandem with the CD3 zeta 小道signaling domain for impr著請oving T cell prolife麗來ration and persistenc化些e.
The structure of CAR has evo做南lved over the past few years based o煙筆n modifications to the com話熱position of the i上山ntracellular domains. The f月空irst-generation CARs consisted雨業 of only a single intr很匠acellular CD3 zeta-derived sig舞站naling domain. While these睡很 CARs could activa這可te T cells, they exh得畫ibited poor anti-tum家女or activity in vivo 風舊due to the low cytotoxicity and外秒 proliferation of T c影木ells expressing麗術 such CARs. This led to the ad不明vent of the secon日年d-generation CARs which included an 答國intracellular costimulato去對ry domain in addition睡金 to the CD3 zeta signaling domain l弟木eading to a significant improvem外人ent in the in vivo商體 proliferation, expansion a匠女nd persistence of 事音T cells expressing second gen熱國eration CARs. To further路員 optimize the ant友呢i-tumor efficacy of C是關AR-T cells, third generation CAR服少s were developed which included two如場 intracellular, cis-acting costimula農我tory domains in是跳 addition to CD3 zeta. Thereafter, fou拍裡rth generation CARs 快喝were derived fr紅林om second-generation CARs by m讀坐odifying their i懂購ntracellular domain for indu會光cible or consti頻窗tutive expression of cytokines.農樂 The fifth and the latest generation 會文of CARs are also derived fr聽到om second-generation CARs by the inco理長rporation of intracellular農又 domains of cytokine recept到明ors.
Our MMLV retrovirus C開線AR expression ve廠身ctor is derived from the Moloney 綠信murine leukemia virus,光呢 which is a member of the retrovirus fa答師mily and is highly sui爸吧table for retrovirus-m大南ediated delivery of second-頻匠generation CAR expression cassett土城es into T cells.
MMLV, a retroviral vecto間制r derived from Moloney murine leuk為鐵emia virus, is a pl她錢us-strand linear RNA vir就美us that exhibits efficient genomic inte化數gration. While our wildtype MMLV retrov議如irus expression vector utiliz煙又es the ubiquitous p用慢romoter function in the 5' lo藍我ng terminal repeat (LTR) of wildt謝林ype MMLV genome for dr技頻iving expression o厭明f the CAR cassette, t兵下he self-inactivating MMLV retrov花照irus expression vector allows users 樂視to select any promoter of their ch城公oice for driving CA年美R expression. This is a技個chieved by the deletion of 下亮the U3 region in the MMLV 3相匠’ LTR which self拍影-inactivates the promoter activity i遠場n the 5' LTR by a都亮 copying mechanism during viral gen話雪ome integration. Th但票is not only prov睡門ides users with the兒街 flexibility to add their 離紅promoter of cho草愛ice for driving CAR expression 科船but also eliminates the risk of oncoge關光nic activation of adjacent g近微enes upon vector integration, thereby 北木enabling such vectors to 家員have a higher safety書還 profile compared to wil學工dtype MMLV vectors.
The self-inactivating MMLV刀制 retrovirus CAR express身坐ion vector is first construct低山ed as a plasmid in E. coli where the醫場 entire CAR expression cas西劇sette including the scFv region, hing吃森e, transmembrane domain and in為信tracellular CD3 z是子eta signaling domain as well as th要麗e costimulatory domai務兵n is cloned in between the two 歌下MMLV LTRs. It is then 厭鐘transfected into packaging cells alon拍上g with several helper plasmi章商ds. Inside the packaging cel影師ls, vector DNA located bet務店ween the two LTRs is transcri年放bed into RNA, and viral proteins expres綠們sed by the helper p師林lasmids further嗎西 package the RNA into viru睡舊s. Live virus is then rel小書eased into the supernatant, which醫在 can be used to infect target北南 cells directly章些 or after concentration. When the 綠西virus is added to target cells,數錯 the RNA cargo is shuttled into 件購cells where it is 火我reverse transcribed into DNA and業個 randomly integrated in th得答e host genome. Any用好 gene(s) that were placed in-betw音村een the two LTR花有s during vector cloning are perman爸南ently inserted into hos場呢t DNA alongside the res身放t of viral genome.
By design, self-inactiva相司ting MMLV retroviral vectors lack街店 the genes required f明又or viral packag爸跳ing and transduct月離ion (these genes are carried by help廠頻er plasmids or 區他integrated into packaging cell科分s instead). As a result, viruses prod間聽uced from these vectors have the im和光portant safety feature o光不f being replicatio門話n incompetent (meaning that they can生遠 transduce target cells but cannot re都人plicate in them).
For further information about th影那is vector system, please refer to th如還e papers below.
| References | Topic |
|---|---|
| Br J Cancer. 120:26 (20森自19) | Review on next-generation CAR明兒 T cells |
| Mol Ther Oncolytics. 3:16014 年關(2016) | Review on CAR models |
| J Immunother. 32:169水不 (2009) | MMLV retrovirus-mediated CAR express自冷ion for autologous adop也離tive cell therapy |
| J Immunother. 32:689 (2009) | Construction and pre-clinical evaluati火區on of an anti-CD19 CAR |
| Mol Ther. 11:1919 (2009) | Insertional tran近舞sformation of HSCs by SIN 熱麗MMLV |
亮點
Our SIN MMLV retr樹綠ovirus CAR expres他少sion vector can be used for the樂鐘 expression of second-generation CARs雪大. It is optimized for high copy num得雜ber replication in E. coli, high-titer媽小 packaging of live virus, efficient銀志 viral transduction of a wide r飛如ange of cells, effi會如cient vector integ雨新ration into the host genome, and hi光房gh-level transgene expression.
優勢
Permanent integration of vector制我 DNA: Conventional 問友transfection results in almost entire風為ly transient delivery of DNA into hos知行t cells due to the loss of DN討城A over time. This pr喝子oblem is especially prominent in 女門rapidly dividing c從可ells. In contrast, retroviral transduct門那ion can deliver genes permanently in在森to host cells due知醫 to integration of the vi但體ral vector into the host開電 genome, thereby enabling long-term ex關作pression of CAR ex鐵金pression cassettes.
Broad tropism: Our packaging system adds th舞藍e VSV-G envelop風紙 protein to the viral 厭到surface. This protein h習現as broad tropism. As a result, ce科紙lls from all commonly used mammal東費ian species such as human, mouse and ra制煙t can be transduced. Fur近動thermore, many sp厭分ecific cell types can be transdu秒愛ced, though our vector has dif兵喝ficulty transducing non-dividing cells 火空(see disadvantages below).
Customizable internal pr通坐omoter: Our vector is d作北esigned to self-in人一activate the promoter 湖聽activity in its 5' LTR呢姐 upon integration i如如nto the genome. As a result, 喝但users can put in their own prom舞可oter to drive their CAR express到計ion cassette within t能光he vector. This is a distinct advantag器我e over our wildtype MMLV r美爸etrovirus vectors, w但少hich rely on the promoter funct化畫ion of 5' LTR t男兵o drive the ubiquito畫訊us expression.
Relative uniformity of deliv亮跳ery: Generally, viral transducti呢劇on can deliver vectors into cells 現可in a relatively u鐵地niform manner. In contrast, convention生山al transfection of pla制分smid vectors can be highly non-uniform匠爸, with some cells receiving子窗 a lot of copie校舊s while other cells receiving few金人 copies or none.
Effectiveness in vitro and i車村n vivo: While our vector is most區明ly used for in vitro transduction of cu但習ltured cells, it can also be used to tr跳玩ansduce cells in live an兒光imals.
Safety: The safety of our vector i笑但s ensured by two fea街亮tures. One is th玩遠e partitioning of genes required聽暗 for viral pack理相aging and transduction into several he國車lper plasmids; 學光the other is self-inactivation of th美分e promoter activ器做ity in the 5' LTR up海媽on vector integratio錢爸n. As a result, it is essential兵現ly impossible for repl算鐵ication competent virus業時 to emerge during packa服花ging and transduction. The health ris鄉去k of working with術看 our vector is therefore minimal.
不足之處
Moderate viral titer: 樹放; Viral titer行下 from our vector reaches ~107懂秒 TU/ml in the supernata明懂nt of packaging時來 cells without further concentration. T靜拍his is about an order of magnit見鐘ude lower than our lenti美很viral vectors.&nb一習sp;
More limited cargo人村 space than wildtype M機們MLV: The MMLV ret林笑roviral genome is 長哥~8 kb. In our vector, 都個the components necessa店大ry for viral packaging and transduct飛又ion occupy ~2.5 kb, which leaves行物 only ~5.5 kb to朋吃 accommodate the user'吃愛s DNA of intere暗白st, including both the CAR expres有劇sion cassette and t雪南he promoter.
Difficulty transducing non器拍-dividing cells: Our vector has difficulty tra低喝nsducing non-dividing cells.好器
Technical complexity少光: The use of MMLV retroviral喝雜 vectors requires the producti章少on of live virus in packaging cells 看笑followed by the m章費easurement of viral titer. T喝厭hese procedures a會區re technically demanding and time 市見consuming relative to電近 conventional plasm黃上id transfection.
載體關鍵元件
CMV promoter: Human cytomegalovirus immediate ear可現ly promoter. It drives transcrip商地tion of viral RNA路熱 in packaging cells. This RNA i來飛s then packaged into 小門live virus.
MMLV 5' LTR-ΔU3: A deleted version of the MMLV老水 retrovirus 5' long terminal分冷 repeat. In wildtype MMLV事你 retrovirus, 5' LTR and 3' LTR are es頻去sentially identical in s愛算equence. They res錢討ide on two ends of the vir老對al genome and point in 玩做the same direction作一. Upon viral integration, the 3' 動爸LTR sequence is copied onto the銀醫 5' LTR. The LTRs carry bot冷場h promoter and poly西愛adenylation fun費市ction, such that the 5' LTR acts as a 做見promoter to drive 紙件the transcription of t會兒he viral genome, while the 3' LTR act長能s as a polyadenylation 拍睡signal to terminate the商服 upstream transcript. On o近木ur vector, MMLV 5' LTR-ΔU3現司 is deleted for a regi店熱on that is required for 車小the LTR's promoter act區工ivity. This does not affec請玩t the production of vir自兵al RNA during packaging be能煙cause the promoter 國這function is supplemented by the要醫 CMV promoter engineered upstr動短eam of Δ5' LTR.
Ψ plus pack2: MMLV retrovir懂女us packaging signal required for t視動he packaging of vir問窗al RNA into virus.
Promoter: The promoter driving you近機r gene of interest is placed here.
Kozak: Kozak cons暗什ensus sequence. It is placed in fro報信nt of the start codon o他女f the ORF of interest科花 because it is believe河員d to facilitate translat又短ion initiation in eukaryotes.
CD8-leader: Leader signal peptide of T-cell surface腦白 glycoprotein CD8 a個現lpha chain. Directs transpor日嗎t and localization 人北of the protein to the友理 T-cell surface.
scFv: Single chain varia快爸ble fragment derived from a也數 monoclonal antib物你ody of known specificity. Recognizes ce笑街lls in an antigen-spe拿分cific manner.
Hinge: Extracellu離作lar hinge region of 知國the CAR. Connects scFv with th城購e transmembrane region都靜 providing stability and f信學lexibility for effic刀河ient CAR expression 開友and function; enhances efficiency o厭道f tumor recognition; improves expansio說問n of CAR-T cells.
Transmembrane domain: Transmembrane domain 匠快of the CAR. Anchors the CAR to the pl作照asma membrane and bridges the extracell讀我ular hinge as well as 都視antigen recognition domains w生中ith the intracellula老子r signaling region; enhances recep子區tor expression and stabi光裡lity.
Costimulatory doma線請in: Intracellular co黑民stimulatory domain of 些明the CAR. Improves overall surv我和ival, proliferation, and 用信persistence of activated金區 CAR-T cells.
CD3zeta: Intracellular domain o年影f the T cell receptor-CD3ζ chai術討n. Acts as a stimulatory m水水olecule for activating T cell-mediate刀鐘d immune response.
WPRE: Woodchuck hepatitis virus posttransc麗紅riptional regulatory element. It enhanc冷術es viral RNA sta上電bility in packaging cells愛動, leading to higher titer of 技都packaged virus.湖術
MMLV 3' LTR-ΔU3:&nb信可sp;A truncated version of你唱 the MMLV retrovirus 3' l知不ong terminal repeat. This leads to t快黑he self-inactivation跳裡 of the promoter acti可見vity of the 5' LTR upon viral ve短紙ctor integration int要藍o the host genome (due to the fa街南ct that 3' LTR is copied onto 5'公呢 LTR during viral inte新亮gration). The polyadenylation signal土我 contained in MML文為V 3' LTR-ΔU3 se厭鐵rves to terminate門微s all upstream transcripts prod歌輛uced both durin去務g viral packaging and af中拿ter viral integrat兵你ion into the host 老費genome.
SV40 late pA: Simian virus 40 late 腦麗polyadenylation signal. I行生t further facil睡雪itates transcriptional 日件termination after the 3' LTR dur國又ing packaging. This elevates t樹知he level of functional viral RNA in 窗話packaging cells, thus i我吃mproving viral tit照會er.
pUC ori: pUC origin of replication. Plasmids ca匠拍rrying this origin exist in h頻自igh copy numbers in E. coli.
Ampicillin: Ampicillin resistance gene. It allow那熱s the plasmid to be maintain機文ed by ampicillin selection in E. col廠生i.