Many gene delivery experimen科花ts utilize viral vectors for eff事知icient delivery of genetic info村村rmation to targe讀章t cells. To mak刀有e your recombinant virus, plasmids cont他要aining your GOI and the 雜冷instructions for pro章那ducing the virus are propagated i歌問n bacterial cells, 家雪then transfected into packaging cells司會. These cells prod學術uce the live recombinant virus, wh務坐ich is isolated, purified,歌拿 and concentrated. Like most molecular 機數biology adventures, these 現我many steps and many days can l算妹ead to heartache or victory. Viral vec藍拿tor heartache can be due 算路to problems includin城理g design, transfection, or gene to男我xicity. If your particular advent行妹ure ends in low quantiti到黑es of virus, here are some tips th小章at we at VectorBui從體lder have collect西身ed to ensure victor聽裡y.

Titration station

After isolating your vir舊城us from the packaging cells, it 小花is important to determine how much w哥理as collected, called t器街he viral titer. This s問習tep is imperative for ensur分月ing efficient transduct秒低ion of your targe女風t cells. However, there are man相樹y different ways 學這to measure viral titer, and dec愛歌iding which method to use can be compl地銀ex and context depen分行dent. A major diffe低筆rentiating factor is whether y公畫ou need physical tite暗對r (the physical店事 amount), functiona問拿l titer (the level of biologica我新l activity), or bo志暗th. Which method varies 購開depending on the virus used靜頻. The table below compares titration事生 methods for popu聽愛lar viruses.

Each titration approach has a月河dvantages and disad新務vantages, especially depending on書美 the space and resources use生綠d for titration. Differences in mea自理sured titer is 新很a common problem becaus信就e various methods, models木弟, reagents, and equipment長醫 can yield different res從熱ults. Additiona她廠lly, measurement after viral 放林degradation can result in low tit很森er.

The titer checklist

When generating viral vectors in t數線he lab, low titer can be草線 attributed to one of the many線短 upstream steps in producti老能on. Ensuring co這也rrect design of your vectors is imperative: cl用又oning inserts into vect區影ors that are beyond carrying長樂 capacity will decrea校子se viral titer, as wil拍水l using plasmids tha看電t contain >70%跳內 GC content across a hundr懂們ed or more bases.&n雜但bsp;AAV’s inverted t多場erminal repeats (ITRs) are 家筆GC rich and can have errors int暗鐵roduced during 現書replication, which d技水ecreases viral titer. En兵區suring ITR integrit美照y is important for ma我少intaining high tite紙跳rs.

Additionally, researc文冷hers must ensure t書線hat they are usin務靜g appropriate helper plasmids and們知 packaging cells. For inst農制ance, second genera如妹tion lentiviral transfer plasmids can空要not be used with 道雨third generation lentiviral packaging p路讀lasmids. Next, pack了麗aging cells must be tra劇家nsfected with the transfer河鐘 plasmids and any helper 長愛plasmids. 樂員If transfection efficiency is low舞但, for instance bec雜來ause the packaging c知白ells exhibit senescence, then v訊技iral titer will be lo要師w. Finally, harvesting the 訊哥resulting virus must be 村金optimized: the h森道arvesting time point and白湖 conditions&nbs國樹p;(e.g. whether collecting f地子rom the supernat上姐ant, cell pellet, o讀你r both).

Genes that kill

If all steps through transfe了件ction have been optimized 暗下and viral titer is still low,件廠 the problem could lie in what the tr相就ansfer plasmid is carrying. The朋金re are a number of genes which are t作購oxic to packaging地下 cells, e.g. pro-apopt近一otic genes like Bax a匠笑nd GSDME, cell cycle regulators li花鐵ke BABAM2 and NEK1, pr水醫oliferation modulators like F2RL1 and F用那oxn1. When these genes are cloned去身 into transfer plasmids, packagin議書g cells are not able to produce hig錢睡h levels of virus, of是空ten due to cytotoxicity (e.g. in 費謝McClean et al., 時事2009 and Janus et al., 2020). Not我車ably, we and others have con笑數sistently found兒場 low viral titer when using vec樂的tors carrying CRISPRa trans黃數 activators p65/HS友樂F1.

To achieve a high viral海低 titer when using toxic gen線飛es, the most st司冷raight-forward 但有approach is to change不公 the promoter to a we店林aker promoter. In th西紙e table below, an AAV vector carr她山ying Foxn1 produce讀票d low viral titers when using str金土ong promoters to drive Foxn1 黃老expression, but changing to a w子快eaker promoter resulted in titers co光很mparable to control virus contai雪子ning non-toxic EGFP.

Additionally, inducible or 理唱tissue specific pro山又moters can be utilized to keep tran南長sgene expressio通拿n levels low in packagi離拿ng cells. If a strong promot子校er is needed in the transfer plasmi吧高d, then packaging ce睡如lls can be modifie生去d to prevent activity o書得f the toxic gene. For i也時nstance, if RNAi is being utilized, DICER can be in筆輛hibited in packa生公ging cells to prevent shR體睡NA activity, which is a場醫 common strategy for packagin弟讀g vectors containing 能拿multiple shRNAs. There are日通 also methods which r綠自epress transgen技你e expression speci新家fically in the pac一民kaging cells to prevent dama湖亮ge. Finally, a toxic trans醫頻gene may be broken u什吃p across multiple transfer plasmids, as照鄉 can be done to acco金南mmodate larger transgenes w劇的hen using AAV. This approach re答器quires careful design and would be美區 best utilized if other meth制機ods are not possible.

Viral vectors allow f影金or gene delivery to 飛開a variety of cell typ要聽es. However, depending on your app術這lication and your tar鐘書get cells, achieving 腦風a concentration of virus that is h中報igh enough can be difficult. Af路高ter ensuring your vectors a習站re properly designed and your tra笑一nsfection rate is optimized, there are雨好 multiple approaches for increasing v又放iral titer. Adjusting p東山romoters on the transfer plasm森樹id or downstream tar短媽gets in the packaging cells a件如re common strategies for 聽車bypassing the effects of toxic ge我街nes. We can help you op劇麗timize your experiment from design t化樂o packaging using VectorBuilder’s extensive experi要弟ence at every step, 算是ensuring toxic genes don’t stand in yo門到ur way.

The table below lays out 信就the genes that we have found to請畫 consistently produce但亮 low viral titer, which benefit司大 from alternative o新動r inducible promo理能ters.

Sources

Janus P, Toma-Jo子機nik A, Vydra N, Mrow懂藍iec K, Korfanty J, Chadalski M, Wid資的łak P, Dudek K, Pas著廠zek A, Rusin M, Polańska J, Widł商現ak W. Pro-death signaling of cytop女微rotective heat shock fac內快tor 1: upregulati河微on of NOXA leading to a河場poptosis in heat-sensitive cel我校ls. Cell Death Differ. 20秒弟20 Jul;27(7):2280-2292. doi: 10.1038公站/s41418-020-0501-8. Epub 2020 Jan 29.玩森 PMID: 31996779; PMCID: PMC7308270.

McLean JE, Datan E, Mat工新assov D, Zakeri ZF. Lack of Bax 水影prevents influenza A v錯照irus-induced apoptosis and c子金auses diminished viral replication. J V讀紅irol. 2009 Aug;83(16):8233-46. doi: 10.鐵區1128/JVI.02672-08. Epub 2009 Jun 3. 銀金PMID: 19494020; PMC工計ID: PMC2715773.

Sena-Esteves M, Gao G. Titra雜短tion of Lentivirus Vectors. Co業到ld Spring Harb Prot店那oc. 2018 Apr 2;201房船8(4). doi: 10.1城務101/pdb.prot095695. PMID: 制亮29610363.

Tiscornia G, Singer海們 O, Verma IM. P音飛roduction and purification of lent農熱iviral vectors. Nat P問也rotoc. 2006;1(1拿窗):241-5. doi: 10.1038/nprot.2006.3中票7. PMID: 17406239.

Wang F, Cui X, Wang M, Xiao W, Xu R. A近多 reliable and feasible qPCR strate木話gy for titrating AAV vectors. Med Sci鐘微 Monit Basic Res. 2013 Jul 可件5;19:187-93. doi: 10.12659/MSM開在BR.883968. PMID: 23828206; 中子PMCID: PMC370640花服9.

Weaver LS, Kadan MJ. Eval公了uation of adenoviral 暗房vectors by flow cytometry員要. Methods. 2000 Jul;自和21(3):297-312. doi: 10.1006/met這書h.2000.1010. PMID: 10873484.