AAV is emerging as a popular tool 謝數for gene delivery in the lab 腦地and in the clinic but腦少 deciding which serotype t視子o use when first starting your e姐麗xperiment can se訊事em like an overwhel一男ming task. Although a broad b數醫ase of literature 購科exists to help guide電鄉 your decision, certain scenarios e化章xist where you mus在間t go beyond the published data. In thi什煙s guide we will dive into wha唱低t specifically differentiates one火裡 AAV serotype from another, 線美how to best target your cells, and high機數light a VectorBu影用ilder product that can easily 現動help a novice AAV re熱化searcher get started 中可with their AAV proj都靜ect.

What Defines a Serotype?

A serotype refers to the subt道影ype of microorganism that can be c小冷lassified together 可兒based on their surface antigens輛看. In the context of viruses,遠我 like AAV, serotype低購 refers to the variation of t草民he viral capsid proteins which dicta放好te their antigenic properties. The AAV票算 capsid is encoded by the cap gene co微麗nsisting of the ORFs for the structu船離ral proteins VP1, VP2, and VP3廠在; together 60 monomers of the綠東 structural proteins interac從坐t to form the AAV capsid線近.

At the genetic lev信歌el, AAV serotypes share a體在 conserved genom爸站e organization with varying leve站生ls of homology amongst the capsid p國玩roteins. Structurally是微, AAV serotypes exhibit similariti開紙es in their basic capsid architecture:醫學 all capsids adopt a similar i數錯cosahedral struc中明ture containing constant a空有nd variable regions鐵醫.  

The constant regions of the AAV cap玩光sid proteins exhibit high homology an了輛d are highly maintai坐小ned between different serotypes.雨微 These regions play critical ro街音les in assembly and maint子好enance of the AAV ca紙看psid structure and contain function不雪al elements critical for packaging the 草理AAV genome. In contr年玩ast, the variable regions of th能一e AAV capsid exhib們暗it significant 河農sequence variation and a到訊re responsible for the di科飛versity of receptor binding domain少動s, antigenic sites, sur校遠face loops, tissue tropis照遠m, and ultimately the diver坐他sity of AAV serotypes.

Figure 1. Cartoon representation of th爸愛e AAV capsid highlighting th吃這e constant (blue) and variable議明 (yellow) regions.醫少

The variable region o鐵懂f the AAV2 capsid 農熱protein has been well s是謝tudied, specifically its surf湖得ace loop involved in 新舞receptor binding of hepar紙舊an sulfate proteoglycan (HS計錢PG) receptors. Arginine residues in對少 this variable region of the surface 科呢loop mediate the interaction些見 of the capsid with the HSPG recepto化下r. Replacement of this area with th分醫e equivalent sequenc車知e from AAV5 resulted in viral草文 DNA packaging but the particles 知玩were non-infectio通海us. Overall, the capsid variable re現月gions are predominantly responsible 開朋for conferring the unique tropism of e拍女ach individual serotype.&n問讀bsp;

Tissue Specificity and Capsid Evol煙們ution

As discussed ab見拍ove, the variation in th有鄉e capsid proteins了從 confers its differential ability信友 to bind to different cellular rec用個eptors and thus, its sp紅喝ecificity for certain cell types, tissu了作es, and organs. T購店he HSPG receptor is wid技黑ely expressed on the surface of 相街most cell types, an雨相d therefore, AAV2 exhib微票its a relatively broad tropism for 少化a variety of ti白聽ssues including liver, muscle, b湖公rain, kidney, retina, a見市nd pancreas, depending on 明線the route of inj謝吃ection. AAV2 has also been shown村到 to interact with other co-re自區ceptor membrane proteins, 人身such as FGFRs and int輛在egrins to enhance viral attachment an頻動d internalization into 民書cells further d兵來ictating its tropism. In contrast上著 to the AAV2 caps子自id, the AAV9 capsid inte西黃racts with galactose and A音答AV1,5,4, and 6 capsid姐是s interact with sialic acid 舞生proteoglycans as their美理 primary attachments for cellular ent器訊ry. This differential preference頻西 for cellular receptors and音愛 co-receptors dictates the tropism o自現f a given AAV serotype.兵美

Figure 2. Cartoon mechanism of AAV2暗人 receptor binding新技 and internalization.

Due to the nature of the 學就AAV capsid and the variety of sero明哥types that have been 筆喝discovered with different鐘房 tissue tropism, a great deal of effor到山t and research has been put i個店nto the discovery黃裡 of new AAV serot火車ypes that confer immune evasion,飛畫 differential tissue tropism, tis什低sue specificity, and incr商下eased capsid internalization. 很美This can be done through a variety 票做of methods including ra很刀tional design or by s個討creening a large l離唱ibrary of AAV cap人開sids generated via d在白ifferent methods.

An example of rational 內火design of an AAV 機月serotype is the AAV2-QuadYF 老電capsid. The rat靜來ionale behind engineeri雪電ng this serotype was to reduce the術店 susceptibility of AAV2 to ubiquit筆間in-mediated proteasomal degradation 答靜and thus increase the number舞國 of capsids capable of properl兵我y delivering the tr算舞ansgene to nucleus. Thi日這s was done by mut是算ation of four tyrosin相她e (Y) residues on the cap公街sid to phenylalanine (F) res物個idues which maintain就鐵s the capsids’ structure她作 but eliminates the ability o腦亮f the amino acid輛拍 to get ubiquitinated b得劇y cellular machinery. Studies 放數with this serotype have 爸還demonstrated increased cellular tra件暗nsduction efficiency and tra美關nsgene expression.

In contrast to the AAV2-Q一吧uadYF serotype, the AAV-DJ serotype was坐報 developed through AAV ca數不psid library construction and scree作物ning. This library of AAV capsids 窗那was created by s歌東huffling the cap gene from multiple A司家AV serotypes. After the library wa草水s generated, it is 我資then subjected to rounds 筆跳of screening in brain tissue until 購舞a novel serotype was志請 revealed that w亮朋as more specific in inf南體ecting the CNS than parental AA坐說V serotypes. Multi現兒ple methods of AAV lib藍鄉rary generation have been publis可女hed and different screening stra姐信tegies can be employed dependen飛光t on the desired properties sought in 北市a novel AAV capsid.

Finding the Right Serotype For Your Exp務暗eriment 

When first starting an AAV project, i長做t can seem like a da熱麗unting task to decipher all the pub身醫lished literature about the properties坐著 and tropisms o校用f different AAV serotype月一s. When working on 歌妹an unpublished system it我日 may even seem impossible to try 技如and choose the correct sero吃吧type for your e月醫xperiment. One way to assess th唱我e AAV serotype suited for you報土r experiment is 開他by systematically comparing 習金the transduction efficiency of di河海fferent serotypes using expression o吧了f a reporter gene by microscopy or f在鄉low cytometry.

The VectorBuilder AAV Serotype Testing Panel is the only commerciall腦河y available AAV ser司票otype testing kit 吃從that offers you the flexibility to cus什問tomize your AAV panel with 3 or more公有 AAV serotypes that can be tested on用訊 a small scale. Thro弟資ugh a combination of literature s吧車earch and customization of the 街場serotype testing panel, 雪作you can quickly identify the serotype 看女best suited for your new 坐拿AAV project in vitro and in 的算vivo. For more information about how什鐵 to get your AAV experiment start短時ed check out the AAV Serotype Testing Panel webpage.

Sources

Opie SR, Warrington KH Jr, Agbandje-McK不紙enna M, Zolotukhin S, Muzyczka N. I看哥dentification of amino acid residue拿哥s in the capsid proteins of匠那 adeno-associated virus type 2 th購錯at contribute to heparan sulfate proteo海服glycan binding. J Virol. 購空2003 Jun;77(12):6995-70務動06. doi: 10.1128/jvi.77拿河.12.6995-7006.2要冷003. PMID: 1276801匠河8; PMCID: PMC156206. 

Meyer NL, Chapman M知的S. Adeno-associated virus (AAV) cel對舊l entry: structural做好 insights. Trends Microb姐生iol. 2022 May;30(5):432-451. d們費oi: 10.1016/j.tim.2021.09.005. Epub 可黃2021 Oct 25. PMID: 34711462.&頻弟nbsp;

Gray SJ, Blake BL, Criswell HE, N校你icolson SC, Samuls這服ki RJ, McCown TJ, Li W.鄉師 Directed evolution of a novel adeno-as還兒sociated virus (AAV) vector t店用hat crosses the seizure-compromised bl不高ood-brain barrier (BBB). Mol 空裡Ther. 2010 Mar;18(3):570-8. doi: 10.校報1038/mt.2009.292. Epub 2009 Dec 29. 現下Erratum in: Mol Ther. 筆朋2010 May;18(5):10體知54. PMID: 20040913; PMCID: 睡計PMC2831133.