Chimeric Ad5/F35 Adenovirus關算 Gene Expression Vector

Recombinant adenovi行議ral vectors are used for ac理行hieving high levels of tra站通nsgene expression in a wide variety 鐘雨of mammalian cell types, where the村冷 vector remains as epis草醫omal DNA without integrating into the 你鄉host genome. High transduction e藍對fficiency and hi路男gh levels of short-term技讀 gene expression make ade生笑noviral vectors the p樂中referred gene delivery tools for ge吃水ne therapy and vaccine researc吧離h.

Adenoviral vectors are deriv兵就ed from adenovirus, which caus市件es the common cold. 輛就Wildtype adenovirus has 他她a double-stranded linear D裡體NA genome.

While human ade技音novirus serotype 5 (Ad5)器朋 vectors are the most widely us但美ed, a major disadvantag綠們e associated wi分司th such vectors is their de劇跳pendence on the 電請coxsackie and adenovirus receptor (CAR議間) for infecting target cel鐵哥ls. Host cells that completely lack or 得場have insufficient 年媽expression of CAR cannot b他唱e efficiently transduced with Ad5 ve謝報ctors. Ad5/F35 ve他很ctors help overco中影me this limitation by expressing慢兒 a chimeric fibe房間r protein comprised們請 of the knob an員路d shaft derived from adeno城要virus serotype 35 (Ad35) and th匠子e fiber tail derived from Ad5笑月. This enables chimeric Ad5/F35 adenovi路拿ral vectors to readily t看朋ransduce CAR-negative 草亮cells in addition to CAR-positiv對雨e cells, by exploiting the a好海bility of Ad35 fiber protein to attach 城照to target cells via a筆車 non-CAR receptor票現, CD46. The chimeric des如我ign of Ad5/F35 adenovirus has been 票近highly instrumental in expanding秒讀 the tropism of黑吧 adenoviral vectors to 機相cell lines such as h都輛ematopoietic cells, primitive stem c樹文ells, vascular smooth musc但些le cells and CAR-negativ火湖e tumor cells which otherwise exhibit 她下poor transduction 店有efficiency with conven北現tional Ad5 vectors.南們

An adenoviral vector is first constru吧說cted as a plasmid in 門自E. coli. It is then transfected in就問to packaging cells, where the region知銀 of the vector between t我有he two inverted terminal repeats (ITRs城木) is packaged in器好to live virus. When the virus is added還線 to target cells, the DNA carg得弟o is delivered into cells where it e紙化nters the nucleus and remains as episom鐘市al DNA without 場視integration into the host genome. Any 校身gene(s) that were p開這laced in-between the two I又問TRs during vector國科 cloning are introduced into target cel睡制ls along with the rest of viral 一著genome.

By design, adenoviral vectors lack the 物友E1A, E1B and E3 genes (delta E就業1 + delta E3). The first two are requ樂嗎ired for the production of live viru子國s (these two genes are 體還engineered into和音 the genome of packaging cells). As a分機 result, virus produced fr街草om the vectors have the imp議文ortant safety feature of being repli湖家cation incompetent (m道他eaning that they can 廠影transduce target cells but cannot r亮喝eplicate in the河能m).

For further informat務術ion about this vector sys說我tem, please refer to t體樂he papers below.

Expanded tropism: The Ad5/F35 vectors h呢習ave an expanded tropism compared to A水上d5 vectors, specifically問民 for cells lacking or havi校是ng insufficient ex老河pression of CAR. The pres拿女ence of a chime明藍ric fiber protein consis站化ting of the knob and shaft derived 務裡from Ad35 on Ad5/F35 vec費靜tors enables them to infe爸書ct CAR-negative ce線錢lls by utilizing the 那亮CD46 receptor, unlike Ad5 ve畫為ctors. 

Large cargo space: The Ad5/F35 vector has a la坐呢rger cargo carrying capacity compare知愛d to the Ad5 vector due to the pre村從sence of the smaller fiber s化得haft coding sequence deri民雜ved from Ad35 in 費子these vectors. As a result電鐘, our Ad5/F35 vector做愛 has about ~8.2 kb of ca他是rgo space to ac道男commodate the user's DNA of interes樹煙t (such as promoter, ORF線費, and fluorescence marker) which 花說is sufficient for most applications都拿.

Low risk of host genome disruption:器水 Upon transducti國熱on into host cells, adenov河計iral vectors remain as episomal DNA市匠 in the nucleus. The l做呢ack of integration into th喝金e host genome can be a desirable f資錢eature for in vivo human applicat什很ions, as it red體玩uces the risk of host g員黃enome disruption that might le子美ad to cancer.

Very high viral tit麗著er: After our adenoviral vector i老區s transfected into packaging cell用秒s to produce live virus, the virus紅從 can be further amplified to very h聽我igh titer by re-infecting pac雪著kaging cells. This is unli就員ke lentivirus, M藍坐MLV retrovirus, or AAV, wh也一ich cannot be amplified by re-i白笑nfection. When adenovir議也us is obtained through our virus pa錯區ckaging service, titer can照小 reach >10¹² VP/ml.

Effectiveness in vitro and in vivo: Our vector is often used to transduce 體什cells in live animals, but知動 it can also be us年兵ed effectively i民遠n vitro.

Safety: The safety of our vec音朋tor is ensured by the 用醫fact that it lacks gen女藍es essential for virus production 吧生(these genes are engineered into th用有e genome of packaging cells). Virus 電草made from our vector is the校件refore replication incompetent e遠技xcept when it is 哥東used to transduce packaging cells什章.

Non-integration of vector不謝 DNA: The adenoviral genome does not in司麗tegrate into the genome of媽呢 transduced cells. Rather, it ex藍內ists as episomal DNA, which can 鐘自be lost over time, espe友些cially in dividing cells.

Strong immunogenicity: Live virus from adenov高理iral vectors ca關小n elicit strong immune外了 response in anim妹一als, thus limiting ce不東rtain in vivo applications.

Technical complexit有林y: The use of aden姐些oviral vectors 那鄉requires the production o國都f live virus in packagin微的g cells followed 音雨by the measurement 科但of viral titer. These procedures are te影我chnically demanding 草山and time consuming.農現