AAV Non-Coding RNA Exp如工ression Vector

The AAV non-coding中就 RNA expression vector is a highl女了y efficient vehicle f花暗or in vitro and in vivo delivery of non遠喝-coding RNAs of interest. N要師on-coding RNAs include a很暗 wide variety of short (<30大金 nucleotides) and long (>20又低0 nucleotides) functional RNA mo個場lecules such as 路車micro RNAs (miRNAs), small interfe都店ring RNAs (siRNAs), piw到照i-interacting RNAs (piRNAs),購火 small nuclear RNAs (snRNAs), 醫了small nucleolar RNA線笑s (snoRNAs), large intergenic n間工on-coding RNAs (li文麗ncRNAs), intronic long no訊光n-coding RNAs (intronic lncRNAs妹吃), natural antisense transcripts 照間(NATs), enhancer RNAs (eRNAs) 習學and promoter-ass習舞ociated RNAs (PARs), none of which ar音問e translated into prot電草eins, however have bee作公n found to play 票得important roles in many cellular鐘用 processes such as 從跳DNA replication, epigene時機tic regulation, t化生ranscriptional and關畫 post-transcrip報大tional regulation an音爸d translation regulation.

The AAV non-coding RNA expression 綠美vector uses an RNA polymerase 上快II promoter to dr要爸ive the expression of the use雜紙r-selected non-c中輛oding RNA gene. This allow她低s the use of tissue-specific, i東林nducible, or variable-strength pro熱地moters, enabling a 又問variety of experiment中弟al applications. For RNA polyme說刀rase II-mediated transcription, t我理he start site is typically in t又大he 3' region of the promoter while文嗎 the termination 議跳site is within th會家e polyA signal sequence. A秒快s a result, the transcript g拍議enerated from this vecto做醫r does not correspond precisely金鐵 to the selected non-co多影ding RNA gene, bu城光t contains some ad術歌ditional sequences both upstream and do放小wnstream. 

The AAV non-coding RNA expression vec快自tor is first constructed as a p鐵老lasmid in E. coli如離. It is then transfected into packa們玩ging cells along 電購with helper plasmids, w舞器here the region of the vector between t拍看he two inverted terminal 請區repeats (ITRs) is packaged into live vi些妹rus. The non-coding RNA of interest pl事得aced in-between the two IT會如Rs is introduced into target 綠門cells along with the res數都t of viral genom亮地e.

The wild-type A藍動AV genome is a linear single-strand到什ed DNA (ssDNA) 綠林with two ITRs forming a hairpin str海視ucture on each end. It is t新樂herefore also known as ssAAV. I雨長n order to express知機 genes on ssAAV vectors房店 in host cells, the ssDNA ge說我nome needs to first be converted to do答樂uble-stranded DNA (dsDNA) through two 道河pathways: 1) synthesis of seco日兒nd-strand DNA by t事劇he DNA polymerase machinery o視術f host cells using the existing ssDNA 低姐genome as the templa這書te and the 3' ITR as the priming s姐好ite; 2) formation of intermolecular dsD日多NA between the pl計公us- and minus-strand ssAAV genomes. The她離 former pathway is知舊 the dominant one.

AAV genomic DNA forms episomal c東可oncatemers in the host cell nucleus.購光 In non-dividing cells, thes喝器e concatemers can remain for 黃視the life of the host cell場線s. In dividing cells, 為但AAV DNA is lost thr文短ough the dilution effect of cell divis腦但ion, because the episomal DN湖知A does not replica著著te alongside host cell DN討黃A. Random integration of AAV DNA into 爸哥the host genome can occur but 小理is extremely&nb用坐sp;rare. This is desirable i放醫n many gene therapy settings wher土兒e the potential oncogenic effect o愛司f vector integration得身 can pose a signif船什icant concern.

A major practical adva著花ntage of AAV is that in 機身most cases AAV can森金 be handled in biosafety l草去evel 1 (BSL1) facilities. This i這樹s due to AAV being也多 inherently replication-匠購deficient, producing little or no i票下nflammation, and causing著和 no known human dise熱睡ase. Due to their low immunoge照熱nicity in host organisms, AAV is the 得秒ideal viral vector for紅司 many animal studie見黑s.

Many strains of AAV have been ident弟市ified in nature. They 文得are divided into different seroty務體pes based on different antig化城enicity of the capsid prote可那in on the viral就靜 surface. Different serotypes can rend吧水er the virus with different ti到做ssue tropism (i.e. tissue specific行放ity of infection). When our AAV vec去秒tors are packaged in暗制to virus, different serotypes ca看視n be conferred 還腦to the virus by using d匠月ifferent capsid proteins for慢黃 the packaging. During cloning, ITRs f什但rom AAV2 are used, as this is commo化技n practice in the field and 黑喝does not impact specificity. Packa小報ging helper plasmids in木月clude a Rep/Cap 船放plasmid, containing the replicatio他湖n genes from AAV2 and the capsid prot分暗eins for a chosen 愛的serotype to determi快水ne tropism. The table遠時 below lists different短高 AAV serotypes and their tissue tropi坐話sm.

For further information大什 about this vector 快工system, please refer to the papers 路購below.