哺乳動物CAR表達慢病毒載體

利用嵌合抗原受體 (Chimer校話ic antigen recep有小tor，CAR) 載體生産可識别腫瘤相關抗原的工程T細胞（也微作稱爲CAR T細胞）已成(chéng)爲一種(zhǒng)極富前景的癌症治動紙療工具。在 CAR T細胞免疫療法中，來自坐還患者（自體）或健康供體（同種(zhǒng)異體）的T細胞經(jīng)過(g問黑uò)改造表達CAR。CAR受體可以靶向(xiàng)識别腫瘤細東志胞表面(miàn)抗原，活化T細胞使其發(fā)揮免疫作用以幫助殺傷飛熱腫瘤細胞。

CAR受體的結構由四個主要部分組成(ch機風éng)：（1）一個胞外抗原識别結構域，由已知特異性的抗體的單鏈短土可變區片段 (Single chain variable fragm從但ent，scFv) 組成(chéng)。 scFv促進(jìn)抗原結合，朋短包含一個輕鏈可變區片段和一個特異的單克隆抗體的重鏈片段，兩(liǎng坐河)者通過(guò)一個柔性的肽鏈相連；（2）一個位于市吃胞外的鉸鏈區或間隔區，將(jiāng)scFv與CAR的跨膜結構域連接起(朋鐘qǐ)來，并爲CAR的結構提供靈活性和穩定性；作聽（3）跨膜結構域，將(jiāng) CAR 錨定在質膜上，從而將(jiāng在看)胞外的鉸鏈區和抗原結合結構域同細胞内結構域關聯起(qǐ)來。它在增強受體表達在吧和穩定性方面(miàn)起(qǐ)著(zhe身鐵)關鍵作用；（4）胞内信号域，通常源自T細胞受體的CD3 zeta 鏈并刀秒包含免疫受體酪氨酸激活基序（Immunoreceptor tyrosi理學ne-based activation motif，ITAM著銀)。抗原結合CAR後(hòu)，ITAM被(b又他èi)磷酸化并激活下遊信号傳導，從熱很而導緻T細胞的後(hòu)續活化。此外，該胞内結構域還(hái)可以引入一個或山計多個與CD3 zeta信号域串聯的共刺激域（來源于CD28、CD137等從筆），以改善T細胞增殖效率和活化的持久性的技。

CAR的結構在過(guò)去幾年中随著(zhe)對(duì)胞内結光街構域的修改而不斷發(fā)展。第一代CAR僅包含一個衍生于CD3 ze讀村ta鏈的信号域。雖然這(zhè)種(zhǒng)CAR可以激長路活T細胞，但是由于表達此類CAR的T細胞的細胞毒性和增殖能(néng)力偏低，有習它們在體内的抗腫瘤活性并不理想。第二代CAR除了包含一個CD歌光3 zeta信号域，還(hái)具有一個胞商大内的共刺激域，因此表達第二代CAR的T細胞顯著改善了其體内增殖能(néng)力大北、擴增能(néng)力和活化的持久性。爲了進(jìn)一步優化CAR 件到T細胞的抗腫瘤功效，第三代CA技她R除了CD3 zeta外，還(hái)引入了兩(li農司ǎng)個胞内順式作用的共刺激結構域。此後(hòu)，通過(g子對uò)修飾胞内結構域以進(jìn)行誘導表達多日或組成(chéng)型表達細胞因子，第四代CAR從第二代CAR開(kāi)發(問土fā)獲得。第五代和最新一代CAR也是從第二代CA南舞R衍生而來，整合了細胞因子受體的胞内結構域。

我們的CAR表達慢病毒載體可用于生産慢病毒并高效地看和向(xiàng)T細胞轉導CAR表達盒。CAR表達慢病毒載體首先在刀在大腸杆菌中進(jìn)行克隆，其中CAR表達盒包含scFV結構冷聽域、鉸鏈區、跨膜區和胞内CD3 zeta信号源、共刺激域，并位于慢病毒載體購綠的兩(liǎng)個長(cháng)末端重複序列（Long termina又光l repeats，LTR）之間。慢病毒載體構建完成(chéng)後訊可(hòu)與輔助質粒一起(qǐ)轉染進快物(jìn)入包裝細胞。在包裝細胞中，位于兩(月商liǎng)個LTR之間的DNA片段會(huì廠市)被(bèi)轉錄成(chéng)RNA，由一劇輔助質粒表達的病毒蛋白將(jiāng)其包裝形成(chén去黑g)病毒顆粒。包裝後(hòu)的活體病毒將西外(jiāng)會(huì)被(bèi)釋放到窗如上清液中，可以直接收集或進(jìn)一步濃縮病毒飛河轉染靶細胞。

當病毒轉導靶細胞時(shí)，釋放到照國宿主細胞中的病毒RNA借助逆轉錄酶逆轉錄成(chéng)雙鏈DNA，然後(h他小òu)随機整合進(jìn)宿主細胞的基因組中。在病毒載用風體中，位于兩(liǎng)個LTR的DNA片就數段和病毒基因組都(dōu)會(huì慢視)穩定整合到靶細胞的基因組中。

通過(guò)改造優化，我們的慢病毒載體删除了與病毒包裝和轉導相關的基因（這湖數(zhè)些基因由輔助質粒進(jìn)行表達，用于病姐算毒包裝過(guò)程），使産生的慢病毒顆粒是複費開制缺陷型的。即包裝的病毒隻具有轉導靶細胞的能(néng)力，而無法在靶細男腦胞中進(jìn)行大量複制，因而具有很高鄉服的生物安全性。

關于該載體系統的更多信息，請參考以下文獻。

RSV promoter: Rous sarcoma virus promoter. It dri司區ves transcription of viral RNA in 呢會packaging cells. This RNA is then pac吧答kaged into live virus.

5' LTR-ΔU3: A deleted version of the HIV-1 5'機歌 long terminal repeat. In wildtype l服購entivirus, 5' LTR and 3' LTR are 開又essentially identic議刀al in sequence. They reside on空業 two ends of the viral genome and point但愛 in the same direction. Upon viral inte自明gration, the 3' LTR seque黑科nce is copied onto the 5'票南 LTR. The LTRs carry both promot光生er and polyadenylation function, s理草uch that in wildtype virus, t化說he 5' LTR acts as a promoter to drive t車員he transcription of the viral gen海和ome, while the 拿會3' LTR acts as a polyaden哥新ylation signal to termi會做nate the upstream transcript. On our ve從了ctor, 5' LTR-ΔU3 is deleted for 林草a region that is re頻事quired for the LTR's p制東romoter activity normally facilit山亮ated by the viral transcription factor中刀 Tat. This does not affect the prod作草uction of viral RNA during packaging 物為because the promoter function is s去物upplemented by the RSV promoter engine吧金ered upstream of Δ5' L視章TR.

Ψ: HIV-1 packaging signal re什答quired for the pac時房kaging of viral RNA into vi河能rus.

RRE: HIV-1 Rev respon又一se element. It allows the 國聽nuclear export of viral RNA機志 by the viral Rev火睡 protein during viral packaging.

cPPT: HIV-1 central polypurine tr我拍act. It creates a "DNA flap" tha白計t increases nuclear importation of市看 the viral genome during target cell 麗銀infection. This improves vector i農謝ntegration into the host g雪光enome, resulting in higher t得醫ransduction effici個民ency.

Promoter: The promoter driving your downstrea少數m CAR expression cassette is pla場知ced here. 

Kozak: Kozak consensus sequence. It i離著s placed in fro南中nt of the start c窗呢odon of the ORF of i答靜nterest because it is believ著木ed to facilitate translation 村船initiation in eukaryotes.

CD8-leader:&nbs做樂p;Leader signal peptide of T cell surfac放不e glycoprotein CD8 alpha chain. Dire日鐘cts transport and localization of the 兵們protein to the T c湖放ell surface. 

scFv: Single chain varia土民ble fragment derived from a monocl相街onal antibody of known specif來我icity. Recognizes cells in 個地an antigen-specific manner. 購子

Hinge: Extracellular hinge region of the CAR.子知 Connects scFv wi用線th the transmembrane region 些計providing stability and flexibility f路文or efficient CAR express民放ion and function; en道新hances efficiency of tumor recogni姐外tion; improves expan服吃sion of CAR T cells. 

Transmembrane domain:&n理腦bsp;Transmembrane domain of the 聽訊CAR. Anchors the CAR to the plasma mem我學brane and bridges the extracellu購那lar hinge as well as a從海ntigen recognition doma用她ins with the intr算鄉acellular signaling region; enhan匠路ces receptor expression and stability.&麗吧nbsp;

Costimulatory domain: Intracellular costimulatory domai下影n of the CAR. Improv購近es overall survi音煙val, proliferation, and persis上司tence of activated書銀 CAR T cells. 

CD3zeta: Intracellular domain of the T cell re店但ceptor-CD3ζ chain. Acts as a stimulato銀那ry molecule for activating T cell-m了議ediated immune response. 

WPRE: Woodchuck hepatitis virus p嗎花osttranscriptional 裡我regulatory element. It enh呢姐ances viral RNA stabi們通lity in packaging cells, leading校草 to higher titer of packaged vir花愛us.

mPGK promoter: Mouse phosphoglycerate遠樂 kinase 1 gene promoter. It dri音你ves the ubiquito來很us expression the down舊近stream marker gene.

Marker: A drug selection gene (such as看刀 neomycin resistance和到), a visually detectable gene (such a近分s EGFP), or a dual生了-reporter gene (suc海雪h as EGFP/Neo). This allows cells t友了ransduced with t頻聽he vector to be selected 唱志and/or visualize有小d.

3' LTR-ΔU3: A truncated version of t就低he HIV-1 3' long terminal 南冷repeat that deletes the歌木 U3 region. This lead學木s to the self-inactivation of the pro通年moter activity of the 嗎外5' LTR upon viral vect器村or integration into知冷 the host genome (due to城些 the fact that 3' LTR is copied東雪 onto 5' LTR during viral inte會紙gration). The polyadenylation signal 票那contained in 3' LTR-ΔU3 ser影畫ves to terminates a多子ll upstream transcripts produc數刀ed both during viral packaging and aft商也er viral integration into t黑民he host genome.

SV40 early pA: Simian virus 40 ear多些ly polyadenylation signal. It體又 further facilitates transcr山匠iptional termination after the 3' LTR 術看during viral RNA transcrip女愛tion during packaging. This elevate兵費s the level of functional亮議 viral RNA in packaging cells,但裡 thus improving viral titer.

Ampicillin: Ampicillin resistance gen路紙e. It allows the pl音姐asmid to be maintained by ampicill哥就in selection in 文答E. coli.

pUC ori: pUC origin of repli公學cation. Plasmids 玩畫carrying this origin exis南商t in high copy numbers們從 in E. coli.