Gutless Adenovirus Gene Expression Ve拍車ctor

Gutless adenoviral vectors (a.k.a. help裡道er-dependent adenoviral vectors) are th自請e latest generation of adenoviral ve新理ctors with a significantly improved s音船afety profile compared to their earl還遠ier generations. The abs讀對ence of nearly all viral seque妹生nces on these vectors except ci光動s-acting elements essential for viral r樂上eplication and packa著跳ging enables gutless aden笑低oviral vectors to exhibi校鐵t minimal immunogenici說短ty and prolonged trans外體gene expression when us唱海ed in vivo, the放錢reby making them highly attract嗎照ive candidates for gene therap算制y. Additionally, 術費the lack of vira愛區l coding sequenc快音es allows them to hav南船e an increased c腦輛argo carrying capacity of up to 37 長妹Kb, making them suitable for expression水問 of long or mult水飛iple transgenes.

Adenoviral vectors are derived from ade購廠novirus, which causes the com們物mon cold. Wildtype adenovirus has 地對a double-stranded lin東呢ear DNA genome.

Since the gutless adeno熱微virus gene expression vector is行知 devoid of all viral codin放場g sequences, viral prot弟短eins required for successf嗎看ul packaging of recombinant virus are s個制upplied in tran暗車s by a helper v姐月irus. An expres快知sion cassette containing the use知下r-selected gene of i黃睡nterest (GOI) driven by a promoter相了 is first cloned了但 into our gutless ade視土novirus vector 器月in between the two ITRs. Add紙火itionally, our vect刀著or incorporates stuf動腦fer sequences of approp算國riate lengths for m街麗aintaining a fi公購nal size of above 28 kb between t用行he two inverted terminal re家樹peats (ITRs), w村校hich is necessary 很近to facilitate the efficient pac北下kaging of virio文亮ns. The region of the v民要ector flanked by th愛都e two ITRs is released from the plas年道mid by restriction digestion. The r得船eleased fragment is tra通離nsfected into packaging cel他睡ls which are subsequently infected wi舞生th the helper virus to 業白generate recombinant adenoviral part月音icles.   

Our gutless adenoviral vector s術嗎ystem utilizes a he森坐lper virus with the packaging s微看ignal flanked by two LoxP sit用水es along with packaging cells w現票hich stably express 鐘微Cre recombinase to facilitate Cre-m拍長ediated excisi件到on of the helper 自笑virus packaging signal. This prevents t師雪he helper virus genome錯紅 from being pac服林kaged into viral parti化美cles along with gutless就算 adenoviral genome. When the recombin好鄉ant virus is added to target 麗厭cells, the DNA cargo is delivered int船廠o cells where it enters the n聽間ucleus and remains as episomal DNA木家 without integration into the h雜上ost genome. Any gene(s) that were p姐刀laced in-between the two I問小TRs during vector cloning are 間去introduced into target cells.

For further information about this vec司黑tor system, please refer t放海o the papers below.

High safety: Our gutless adenoviral vectors are c去家haracterized by the absence 輛討of all viral seq器理uences except cis-acting elements es放笑sential for viral re站用plication and packaging, leading這線 to a significant improvement in 人廠their safety profil不票e compared to the previou朋科s generations o湖草f adenoviral vectors.懂子 As a result, such vectors offer生書 the advantages of min志身imized host immune respons們資e and prolonged transgene expre家好ssion in vivo.

Low risk of host gen紙但ome disruption: Upon transduction into host ce什下lls, adenoviral vectors r少間emain as episomal DNA in t物有he nucleus. The l計紙ack of integration into the host可了 genome can be a desirable feature for 行美in vivo human applications, as i關如t reduces the r對為isk of host genome disruption that mi窗明ght lead to cancer.

Very high viral titer: After our adenov線裡iral vector is tr銀樹ansfected into packaging cells to pro舊老duce live virus, the v會道irus can be furt城玩her amplified to very high titer b內算y re-infecting p遠個ackaging cells. Thi畫術s is unlike lentiv校空irus, MMLV retrovirus, or AA草慢V, which cannot be amplifi學雪ed by re-infection. When ade紙刀novirus is obtain吃老ed through our virus 機水packaging service, titer can 物作reach >10¹⁰ infectious units麗能 per ml (IFU/ml).

Broad tropism: Cells from commonly used m校答ammalian species such as human, mous明物e and rat can be transduced with our ve窗紅ctor, but some cell typ技這es have proven difficult to transduc美紅e (see disadvantages below)到生.

Large cargo space: The deletion of viral coding sequ個如ences renders gutless aden空呢oviral vectors with a l文讀arge cargo carrying capacity報男 of up to 37 kb, 讀紅making them highly suitable for applica劇行tions requiring expressi的草on of large or multiple transge林樹nes.

Effectiveness in vitro and i商呢n vivo: Our vector is often used to transduc謝校e cells in live animals, but it ca地到n also be used effectively in v高拍itro.

Non-integration of vector DNA: The adenoviral genome does not in雜笑tegrate into the genome 些車of transduced cells. Rather, it exists 風還as episomal DNA, which can be lost ov湖報er time, especially in dividing cel離著ls.

Difficulty trans費月ducing certain ce動有ll types: While our adenoviral ve工黃ctors can transduce 場長many different cell信大 types including non-div北舞iding cells, it is inefficien市廠t against certain cell typ離商es such as endothelia, smooth m山廠uscle, differentiated airw現爸ay epithelia, peripher短錢al blood cells, neurons, and hema靜新topoietic cells.

Helper virus con可雜tamination: Our gutless adenoviral vector男化 system utilizes河了 a helper virus containing a flo費科xed packaging signal along with pac術你kaging cells which stably express Cr用視e recombinase to restrict t火信he helper virus genome from bein紙地g packaged into viral particl化船es along with the就票 gutless adenoviral genome b很去y Cre-mediated excision of the helper 開明virus packaging signal. How市兵ever, low levels of helper virus con書城tamination might still be present i我金n the recombinant viral preps 雪不due to inefficient excis麗科ion of the packaging s間我ignal. The presence of such low leve但中ls of helper virus contamination might 中工have associated toxicity effects a車有t high doses of th學月e recombinant virus但花.  

Technical complexity: The production and amplification 上舊of recombinant gutless 務大adenoviral vectors is far more tec務不hnically challenging compare兵河d to the earlier房問 adenoviral vect行紙or generations because it involve內路s the addition of the h些月elper virus with each pas對件sage.