Adeno-Associated Vi呢黑rus Gene Expression Vector (sc說黃AAV)

The adeno-associated v算校irus (AAV) vector system科多 is a popular and versatile 筆身tool for in vitro and in vivo gen匠機e delivery. AAV is effective in transd著廠ucing many mammalian cell types, and, 東聽unlike adenovirus, has very low作自 immunogenicity, being almost entirel快我y nonpathogenic in vivo. Th舞媽is makes AAV the ideal viral 資文vector system fo舞文r many animal studies. 

An scAAV vector 船小is first constructed as a plasmid in個著 E. coli. It is the生廠n transfected into pack筆坐aging cells along with helper plasmi影妹ds, where the region of the vector好中 between the two購通 inverted terminal repea話場ts (ITRs) is packaged into live viru身跳s. Any gene(s) placed in-between the tw車銀o ITRs are introduced into targ區器et cells along with分子 the rest of viral genome.

The wild-type AAV genome is a linear si木來ngle-stranded DNA (ssDNA) with two I靜不TRs forming a hairpin通拿 structure on each end. 區志It is therefore also 民制known as ssAAV. In 南內order to express genes on ssAAV vect習問ors in host cells, the ssDNA genome nee日國ds to first be conv請對erted to double-stranded DNA (ds綠裡DNA) through two pathways: 1) s他木ynthesis of sec好慢ond-strand DNA by the DNA 從舊polymerase machinery of h朋見ost cells using the exis坐電ting ssDNA genome as the template and 火空the 3' ITR as the priming site物唱; 2) formation of intermolecular 城服dsDNA between the plus- and子雨 minus-strand ssAAV genomes. The fo家現rmer pathway is th理黑e dominant one.

Our scAAV transfer vector is eng影嗎ineered from ssAAV w光木ith two important differences. Fi麗短rst, the trs (terminal res公冷olution site) located in the 3森到' ITR is deleted in 黑習scAAV. As a resu算開lt, scAAV has a tendency of forming a 師話single-stranded DNA molecule黑頻 during replication that is th我區e concatenation of two的吃 full single-stranded geno他好mes, one plus strand and 能讀the other minus st那少rand. This molecule can form a self-c腦子omplementary intramolecular dsDNA gen熱會ome. When scAAV viral particles enter 拍腦host cells, this self-complementary i明區ntramolecular dsDNA ge還好nome can skip second-strand synthes黃中is, which is the議司 main rate-limiting step 行刀associated with conventional ss要遠AAV transduction, 放微to quickly express genes城放 carried on the scAAV這得 vector. Therefore, scAAV has fa快司ster and increased transgene expr見服ession relative to ssAAV. Second, due 上生to fact that wildtype AAV c厭林an carry up to 裡關about 4.7 kb of single-stranded DNA照時 genome and yet 內他each scAAV DNA mol外微ecule packaged into a viral pa高劇rticle is the concat們理emer of two single-stranded ge照會nomes of opposite strands, the cargo要員 capacity of scAAV in terms of t睡年he length of the 5' ITR to 內歌3' ITR transgene that can be醫愛 properly packaged into matu也我re virus is only about half理的 that of ssAAV.

A major practical a了離dvantage of AAV is that in mo數著st cases AAV can b弟學e handled in biosafety level 1 (B電服SL1) facilities. This is due to AAV b員東eing inherently re區間plication-deficie河但nt, producing little or no i愛門nflammation, and causing no k業玩nown human disea姐身se.

Many strains of AAV have been identifi機也ed in nature. They are divided into d友聽ifferent serotypes based on d光件ifferent antigenici樂視ty of the capsid protein on the v從黑iral surface. Differ但技ent serotypes c兒房an render the virus with d業林ifferent tissue tropism (i.e. 樂這tissue specificity of infection). When 笑歌our AAV vectors 下哥are packaged into virus, different sero錯友types can be conferred to the viru相好s by using different capsid proteins對答 for the packaging. The s路算erotypes currently 我來offered by us for our ssAAV and scAA村時V vector systems include近林 - serotypes 1, 2, 3, 4, 5, 6, 6.2, 7內路, 8, 9, rh10, DJ, DJ/8, 為門PHP.eB, PHP.S, AAV2-空理retro and AAV2-QuadYF. During c音生loning, ITRs from AAV2 are u還坐sed, as this is common practic農會e in the field and does not impact spe要爸cificity. Packaging helper plasmids i紙老nclude a Rep/Cap plas請書mid, containing the replic影外ation genes from AAV2 and間離 the capsid proteins雨票 for a chosen serotype to det行唱ermine tropism. The 就西table below lists diffe林區rent AAV serotypes and their tissue tr靜男opism. 

For further information about 南火this vector system, 畫金please refer to the papers b林女elow.