Burada sunulan çalışma Sağlık Araştırması (CIHR) Kanada Enstitüleri tarafından desteklenmiştir (DCB-120266, PPP-133377 ve AG için HBF-348967 verir).

1. Hücre ve Transfeksiyonlar <ol><li> Dulbecco&#39;nun modifiye Eagle ortamında (DMEM) içinde kültür HEK 293T hücreleri,% 10 cenin sığır serumu (FBS) ve% 1 penisilin / streptomisin ile takviye edilmiştir. Hücre kültürü ortamı içinde 2 x 10 5 hücre / ml&#39;lik bir süspansiyon hazırlayın. Her çukuruna hücre süspansiyonundan 500, 100 ve 1000 ul, 24 oyuklu, 96 gözlü, 12 gözlü levhalar, viral üretimi, hücre canlılığı ve immün aktivasyon deneyleri sırasıyla (Şekil 2A) için. </li><li> Yavaşça girdap plakalar ve% 5 CO 2 ile 37 ° C&#39;de / Ç N onları kuluçkaya yatmaktadır. % 50-70 confluency hücreleri büyür. </li><li> Bir transfeksiyon planına göre, 1.5 ml mikro tüpler (örneğin Şekil 2B) test RNA&#39;lar ve kontrol seyreltileri hazırlanır. <ol><li> Viral üretimi deneyi için, bir HIV-1 ifade plazmidinin 10 ng / | il seyreltme hazırlanması ve her bir tüpe 10 ul ekle. Sonraki 5 uM test RNA&#39;lar dilüsyonları ve negatif kontrol RN hazırlamakC. 25 ya da 100 nM nihai konsantrasyonlarda, ilgili tüplere her bir test RNA ve negatif kontrol seyreltme 2.5 ya da 10 ul ekle. </li><li> C: Hücre canlılığı deneyi için 5 uM test RNA dilüsyonları ve pozitif kontrol RNA, düşük molekül ağırlıklı poli I bir 10 mg / ml seyreltme hazırlar. sırasıyla, 100 nM veya 200 ug / ml nihai konsantrasyonları, tekabül eden tüpler test RNA ya da pozitif kontrol RNA dilüsyonları 2 ul ekle. </li><li> immün aktivasyon deneyi için, deney RNA veya adım 1.3.2 hazırlanan pozitif kontrol RNA dilüsyonları 20 ul ekle. sırasıyla, 100 nM veya 200 ug / ml nihai konsantrasyon, tekabül eden tüplere. Aşama 1.3.1 ng 25 ve 100 nihai miktarda vererek, test RNA ekspresyon plazmidleri, deney RNA&#39;lar 5 uM dilüsyonları yerine 10 ng / | il dilüsyonları hazırlamak: edin. 100 adım 1.3.2 ng. ve 1.3.3. </li></ol></li><li> Viral ürünler için, her transfeksiyon tüpüne DMEM 50, 25 ya da 75 ul eklesırasıyla uction, hücre canlılığı ve bağışıklık aktivasyon tahlilleri. Viral üretim deneyleri için, bir sonraki adımdan önce bir biyo-güvenlik seviyesi 3 (BSL3) laboratuvara hücreleri ve hazırlanan transfeksiyon tüpleri getirmek. </li><li> transfeksiyon tüplerine transfeksiyon reaktif sırayla 2 ul ekleyin ve kompleksleri oluşturmak için izin vermek için 15 ila 20 dakika inkübe edin. Belirli transfeksiyon reaktif Malzemesi Tabloya bakınız kullanılacak. </li><li> damla damla hücre kültür plakaları içinde karşılık gelen pozisyonlarda, her mikro tüp tamamını transfeksiyon karışımı ekleyin. Yavaşça girdap ve% 5 CO2 ile 37 ° C &#39;de 48 saat süre ile inkübe edin. </li><li> Hücre kültürü plakaları (Şekil 2C), HIV-1 üretimini (Bölüm 2), hücre canlılığı (bölüm 3) ve immün aktivasyon (bölüm 4) ölçün. </li></ol> 2. Viral Üretim Deneyi <ol><li> inkübatör 24-iyi hücre kültür plakaları çıkarın ve BSL-3 hücre kültürü içindeki girdap plakalar hafifçedavlumbaz. HIV-1 üretimini ölçmek için kullanılacak olan 96-gözenekli düz tabanlı plaka içinde de karşılık gelen her gözden üst fazın 150 ul aktarın. Not: Genel viral miktar deneyleri, enzim-bağlı immünosorbent deneyi (ELISA), 20, ters transkripsiyon polimeraz zincir reaksiyonu (RT-PCR) 21 yoluyla viral RNA miktarının HIV-1 kapsid proteini (p24) ekspresyonunu ölçmek ve aktivitesinin ölçülmesi bulunmaktadır HIV-1 RT enziminin. 2.2 2.5 HIV-1 RT aktivitesinin 13,15,16 ölçmek için yöntemler açıklar Adımları. </li><li> Aktarım, bir viral bozulması kokteyli 15 (Tablo 1) 25 ul ihtiva eden 96 oyuklu bir plaka gözenekleri içinde karşılık gelen süpernatan 5 ul. Oda sıcaklığında 5 dakika süreyle inkübe karışımı ve radyoaktivite iş istasyonuna plaka aktarın. Not: Adım 2.2. Bir radyoaktivite iş istasyonu BSL3 laboratuarda mevcut değilse gereklidir. Plakalar kaldırılması gerekmiyorsaBSL3 laboratuar, 2.3 adıma geçin. ve radyoaktif kokteyl 25 ul yerine viral / radyoaktif bozulması kokteyli (Tablo 1) 50 ul ekle. </li><li> Radyoaktif bir kokteyl 15 (Tablo 1) Hazırlama ve viral süpernatan ve baskıyı kokteyl her çukuruna 25 ul ekle. 2 saat boyunca 37 ° C&#39;de inkübe edin. </li><li> Nokta 5 cam elyaf Di Etil Amino Etil (DEAE) Filtermat kağıdı karşılık gelen kareler üzerine reaksiyon karışımının ul ve noktalar 10 dakika kurumasını bekleyin. hiçbir iki numune doğrudan birbirlerine yatılı böylece her meydanda reaksiyon karışımı nokta. Bu aşamada 2.6 dakikada (cpm) başına sayıları belirlerken örnekler arasında çapraz-over önlemeye yardımcı olur. </li><li> Kağıtlar% 95 etanol ile iki kez 1 dakika yıkama yapılmıştır 2x tuzlu su, sodyum sitrat (SSC) tampon maddesi (Tablo 1) 5 dakika için 5 kat yıkayın. kağıtlar kurumaya ve örnek torbalarına bunları mühürlemek için izin verir. </li><li> Klip örnek çanta coBir kaset içine Filtermat kağıt ntaining ve bir mikroplaka sintilasyon sayacı içine kaset takın. Deneyde kullanılan [32 P] dTTP parti için sağlanan referans tarihi ile 32 P cpm okumak için sayaç ayarlayın. plaka haritayı kullanarak okuma ve sayaç başlatmak için hangi örnekler seçin. </li><li> herhangi bir virüs ölçümü yöntemi için, komşu negatif kontrol ile her test RNA için elde edilen değerler bölmek ve test RNA&#39;ların her tekrarında HIV-1 üretiminin yüzde inhibisyonunu elde etmek için 100 ile bu değeri çarpın. Farklı konsantrasyonlarda farklı test RNA karşılaştırma sonuçlarının bir örneği Şekil 3&#39;te verilmektedir. </li></ol> 3. Hücre Canlılık Testi <ol><li> / Ml MTT (3- [4,5-dimetil-2-tiyazolil] -2,5-difenil-2H-tetrazolyum bromid) Dulbecco fosfat tamponlu tuzlu su içinde seyreltildi (inkübatör, 96 gözlü levhalar çıkarın ve 5 mg 20 ul her bir kuyunun DPBS). f inkübeveya 37 ° C&#39;de 3 saat. </li><li> deterjan (% 1 NP-40, izopropanol içinde 4 mM HCI) her ile asitleştirildi izopropanol 150 ul ilave edin ve oda sıcaklığında 2 saat boyunca inkübe edin. </li><li> Bir mikrolevha spektrofotometre 570 nm&#39;de absorbans belirler. </li><li> bitişik transfeksiyon kontrolü ile her numune için elde edilen değer bölünerek, her pozitif kontrol ve test RNA göreceli MTT metabolizmasını hesaplayın. Farklı test RNA ve pozitif kontrol karşılaştırma sonuçlarının bir örneği Şekil 4&#39;te verilmektedir. </li></ol> 4. Bağışıklık Aktivasyon Testi <ol><li> inkübatör 12-kuyu plakaları kaldırmak ve kültür ortamı aspire. Yavaşça DPBS hücreler iki kez yıkayın ve her bir (proteaz ve fosfataz inhibitörleri, Tablo 1 de dahil olmak üzere), soğuk lisiz tamponu 22 70 ul ekle. Buz üzerinde 10 dakika süreyle inkübe edin. </li><li> mikro tüpler hücre lizatları aktarın ve hızlı daldırarak bunları dondurmaksıvı nitrojen içinde borular. Numuneler Çözülme ve 3 olmak üzere toplam 2 daha donma çözülme döngüleri için tekrar izin verin. </li><li> Hücre yıkıntıları pelet haline getirildi, 4 ° C&#39;de, 15.700 x g&#39;de 15 dakika boyunca santrifüj lizatları. Yeni bir mikro tüpler süpernatantı aktarın ve Coomassie mavisi (Bradford) yöntemini 23,24 kullanarak protein konsantrasyonunu belirleyin. </li><li> % 10 denatüre edici poliakrilamid jel içinde her bir numunenin proteinin 75 ug çözmek ve daha önce 25,26 tarif edildiği gibi bir nitroselüloz zara transfer. </li><li> Bir membrana elektroforezi ve aktarımı sonra, iki kez distile su ile yıkama, ardından 1 dakika boyunca Ponceau S (Tablo 1) &#39;de membran inkübe edilerek protein bantları görülebilir. 80 ve 55 kDa zar kesmek için bir kılavuz olarak bantlar ve protein merdiven kullanın. Not: Adım 4.4 örnekleri 16 x 18 cm 34 kDa aşağı 2 jelleri çalıştırılabilir, belirtilen pozisyonlarda membran kesip kesip değil kolay şekildeilgi bantları. Alternatif olarak, çeşitli jeller zarı kesme önlemek için aynı örnekleri ile çalıştırılabilir. </li><li> % 0.05 Tween 20 (TBST, Tablo 1) ihtiva eden Tris-tamponlu tuzlu su ile Ponceau S boyama yıkayın. Tamamen zarları kapsayan% 5 yağsız süt ile TBST ilave edin. 1 saat boyunca çalkalama ile, oda sıcaklığında membranlar inkübe edin. </li><li> membranlar inkübe O / N ADAR1 (110 ve 150 kDa), fosfo-PKR (62 kDa) ve fosfo-IRF3 (47-kDa) karşı 1,000&#39;de 1 seyreltilmiş% 3 sığır serum albümini ile TBST (BSA) ve antikorlarda, sırasıyla membran, üst orta ve alt parçalar için. </li><li> Membranlar TBST ile 5 dakika için 5 kat ve 1 saat (5,000 içinde 1 seyreltilmiş),% 5 yağsız süt ve peroksidaz-etiketli keçi anti-tavşan sekonder antikor ile TBST içinde inkübe yıkayın. </li><li> TBST ile 5 dakika boyunca membranlar 5x yıkayın ve üreticinin talimatlarına göre filmler bantları görselleştirmek için elektrokemiluminesan (ECL) çözümü uygulayın. </li><li> filmler protein bantları görselleştirmek sonra bir antikor sıyırma çözeltisi ile 10 dakika boyunca zarın, orta ve alt parçaları yıkayın. ve IRF3 (500 &#39;de 1 olarak), toplam PKR karşı% 3 BSA ile TBST içinde membranlar O / N ve antikor inkübe (1.000 de 1), sırasıyla, orta ve alt parçalar için. Yineleyin 4.8 adımları. ve 4.9. PKR membran (orta), anti-tavşan ikincil antikoru yerine peroksidaz etiketli keçi anti-fare kullanılmıştır. </li><li> TBST ile 5 dakika için membran 5x alt parça yıkayın (5,000 içinde 1 seyreltilmiş) aktine karşı bir antikor ile 1 saat boyunca% 3 BSA ile TBST içinde membran inkübe edin. Yineleyin 4.8 adımları. ve 4.9. anti-tavşan sekonder antikor yerine peroksidaz etiketli keçi anti-fare kullanılmıştır. Farklı test RNA ve pozitif kontrol karşılaştırma sonuçlarının bir örneği Şekil 5&#39;te sağlanmaktadır. Kullanılacak özel antikorlar için Malzemelerin tabloya bakınız. </li></ol>

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<li>Burugu, S., Daher, A., Meurs, E. F., Gatignol, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HIV-1+translation+and+its+regulation+by+cellular+factors+PKR+and+PACT%5BTitle%5D)+AND+%22Virus+Res%22%5BJournal%5D)">HIV-1 translation and its regulation by cellular factors PKR and PACT.</a> Virus Res. 193, 65-77 (2014).</li>
<li>ter Brake, O., Konstantinova, P., Ceylan, M., Berkhout, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Silencing+of+HIV-1+with+RNA+interference%3A+a+multiple+shRNA+approach%5BTitle%5D)+AND+%22Mol+Ther%22%5BJournal%5D)">Silencing of HIV-1 with RNA interference: a multiple shRNA approach.</a> Mol Ther. 14, 883-892 (2006).</li>
<li>Naito, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimal+design+and+validation+of+antiviral+siRNA+for+targeting+HIV-1%5BTitle%5D)+AND+%22Retrovirology%22%5BJournal%5D)">Optimal design and validation of antiviral siRNA for targeting HIV-1.</a> Retrovirology. 4, 80(2007).</li>
<li>Mandal, D., Feng, Z., Stoltzfus, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Excessive+RNA+splicing+and+inhibition+of+HIV-1+replication+induced+by+modified+U1+small+nuclear+RNAs%5BTitle%5D)+AND+%22J+Virol%22%5BJournal%5D)">Excessive RNA splicing and inhibition of HIV-1 replication induced by modified U1 small nuclear RNAs.</a> J Virol. 84, 12790-12800 (2010).</li>
<li>Chang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enhanced+expression+and+HIV-1+inhibition+of+chimeric+tRNA%28Lys3%29-ribozymes+under+dual+U6+snRNA+and+tRNA+promoters%5BTitle%5D)+AND+%22Mol+Ther%22%5BJournal%5D)">Enhanced expression and HIV-1 inhibition of chimeric tRNA(Lys3)-ribozymes under dual U6 snRNA and tRNA promoters.</a> Mol Ther. 6, 481-489 (2002).</li>
<li>Chang, L. J., Liu, X., He, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lentiviral+siRNAs+targeting+multiple+highly+conserved+RNA+sequences+of+human+immunodeficiency+virus+type+1%5BTitle%5D)+AND+%22Gene+Ther%22%5BJournal%5D)">Lentiviral siRNAs targeting multiple highly conserved RNA sequences of human immunodeficiency virus type 1.</a> Gene Ther. 12, 1133-1144 (2005).</li>
<li>Daniels, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HIV-1+RRE+RNA+acts+as+an+RNA+silencing+suppressor+by+competing+with+TRBP-bound+siRNAs%5BTitle%5D)+AND+%22RNA+Biol%22%5BJournal%5D)">HIV-1 RRE RNA acts as an RNA silencing suppressor by competing with TRBP-bound siRNAs.</a> RNA Biol. 12, 123-135 (2015).</li>
<li>Castanotto, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Combinatorial+delivery+of+small+interfering+RNAs+reduces+RNAi+efficacy+by+selective+incorporation+into+RISC%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">Combinatorial delivery of small interfering RNAs reduces RNAi efficacy by selective incorporation into RISC.</a> Nucleic Acids Res. 35, 5154-5164 (2007).</li>
<li>Vickers, T. A., Sabripour, M., Crooke, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((U1+adaptors+result+in+reduction+of+multiple+pre-mRNA+species+principally+by+sequestering+U1snRNP%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">U1 adaptors result in reduction of multiple pre-mRNA species principally by sequestering U1snRNP.</a> Nucleic Acids Res. 39, e71(2011).</li>
<li>Tai, C. J., Li, C. L., Tai, C. J., Wang, C. K., Lin, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Early+Viral+Entry+Assays+for+the+Identification+and+Evaluation+of+Antiviral+Compounds%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds.</a> J Vis Exp. , (2015).</li>
<li>Riss, T. L., et al. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aRiss%2C+TL+%22Assay+Guidance+Manual%22">Assay Guidance Manual</a>. Sittampalam, G. S., et al. , (2004).</li>
<li>Reynolds, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induction+of+the+interferon+response+by+siRNA+is+cell+type-+and+duplex+length-dependent%5BTitle%5D)+AND+%22Rna%22%5BJournal%5D)">Induction of the interferon response by siRNA is cell type- and duplex length-dependent.</a> Rna. 12, 988-993 (2006).</li>
<li>Whitehead, K. A., Dahlman, J. E., Langer, R. S., Anderson, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Silencing+or+stimulation%3F+siRNA+delivery+and+the+immune+system%5BTitle%5D)+AND+%22Annu+Rev+Chem+Biomol+Eng%22%5BJournal%5D)">Silencing or stimulation? siRNA delivery and the immune system.</a> Annu Rev Chem Biomol Eng. 2, 77-96 (2011).</li>
</ol>

The authors have nothing to disclose.

Tanımlanan HIV-1 üretimi deneyi HEK293T hücreleri (Şekil 2) kullanılarak gerçekleştirilmiştir ve etkili bir ribozim 13 shRNA 10,29, siRNA 30 ve U1i RNA 11,31 hedef sitesi HIV-1 RNA taramak için kullanılan deneyler benzer edildi. HIV-1 üretimini ölçmek için çeşitli yöntemler kullanılarak, çalışmaların çoğu, aday RNA&#39;lar ile bir HIV-1 ifade plazmidinin Müşterek transfeksiyon sonrasında, virüs üretimini 48 saat ölçtük. HIV-1 ü...

Mevcut HIV-1 terapileri sınırlaması, kronik hastalığın ilerlemesini engellemek için de uygulanabilir olmasıdır. HIV-1 karşı T lenfosit veya hematopoietik kök hücre nakli, ilaç tedavisi 1,2 yokluğunda HIV-1 replikasyonu, uzun süreli kontrolünü sağlama potansiyeline sahiptir ve aynı zamanda, bir HIV-1 ilacı elde etmek için etkili bir yaklaşım olabilir 3.. HIV-1 replikasyonu dirençli hücre oluşturmak için bir yolu, otolog transplant 4 sırasında enfekte bireyin hücrelerine anti-HIV-1 RNA veya peptitler kodlayan bir ya da daha fazla gen eklemektir. Çeşitli aday anti-HIV-1 genlerinin herhangi bir tek genin HIV-1 direnç gelişmesini önlemek için, iki adet 5 ya da üç 6 kombinasyonları bazı giren klinik çalışmalarda dizayn edilmiştir. Anti-HIV-1 RNA immün yanıtları ortaya çıkarmak için düşük potansiyel ve onlar için kombine gen terapisi için en iyi adaylar arasındaÇok kısa bir gen sekanslarından transkribe edilmektedir. Bazı anti-HIV-1 RNA, viral giriş ve bütünleşmesini hedeflemek üzere dizayn edilmiştir. Bununla birlikte, en anti-HIV-1 RNA, viral yaşam çevriminde sonrası entegrasyon adımlar (Şekil 1) hedefler. Sonrası entegrasyon inhibitörleri ribozimler 7, 8 ve shRNAs U1i RNA&#39;lar 9 olarak, HİV-1 düzenleyici proteinleri tat veya rev 1 ve antisens dayalı RNA hedef HIV-1 RNA farklı sitesi hedef, yem RNA içerir. Anti-HIV-1 RNA etkinliğini karşılaştırmak için kullanılmış olan yöntemler genlerin aday RNA&#39;lar kodlayan ve viral geçici aday RNA ifade eden plasmidler ile transfekte edilen hücrelerde üretim ve HIV-1 ifade plazmid ölçüm ile dönüştürülmüş hücrelerin viral replikasyonu izlenmesini, 10 -13. Daha önce yeni bir ribozim, hedef siteye 13-15 HIV-1 RNA taranması için bir HIV-1 üretimi deneyi kullandık. Bu yöntemler beri bir RNA biçimini optimize etmek için rafine edilmişgirişim molekülü, bir shRNA plazmid DNA&#39;sından ifade edilen ya da sentetik bir siRNA 16 olarak verilir. Deney insan embriyonik böbrek (HEK) 293T hücrelerinde olgun virüs üretimini ölçer ve (Şekil 1), HIV-1 replikasyon döngüsünde sonrası entegrasyon evreleri hedefleyen önleyicilerinin etkilerini karşılaştırmak için kullanılabilir. Ön entegrasyon basamakları hedefleyen inhibitörleri, böyle bir tzm-bl hücre enfektivite tahlil 17 gibi alternatif deneyler antiviral etkinliğini değerlendirmek için gereklidir. Klinikte anti-HIV-1 RNA teslimi için önemli güvenlik kaygıları, insan RNA veya protein ve doğuştan gelen bağışıklık sensör aktivasyonu üzerindeki potansiyel hedef dışı etkiler bulunmaktadır. Anti-HIV-1 siRNA&#39;lar toksisitesini değerlendirmek için, farklı hücre hatları 16 bir hücre canlılığı deneyi kullandık. Ayrıca, iki iplikçikli bir RNA bağışıklık sensörler aktivasyonu ölçülmüştür RNA aktive edilmiş protein kinaz R (PKR) reseptör 3 (TLR3) Toll benzeri gibi exinterferon pression geni ADAR1 P150 uyarılmış. Bu deneyler, anti-HIV-1 RNA etkinliği hücre canlılığı veya immün sensör aktivasyon dolaylı etkileri nedeniyle olmadığını teyit etmek için kullanılabilir. Ayrıca, daha da geliştirilmesi, potansiyel toksisite aday RNA&#39;ların hariç yararlıdır. Aşağıdaki protokollerde, prosedürler yeni tedavi RNA&#39;lar belirlemek ve mevcut biçimi açıklanmıştır optimize etmek. Yöntemleri, HIV-1 replikasyonu tarama RNA temelli sonrası entegrasyon inhibitörleri için faydalı olan ve bu tür viral RNA 18 ya da CRISPR Rev aracılı ihracat hedefleme küçük moleküller gibi diğer sonrası entegrasyon inhibitörlerinin taranması için adapte edilebilir / tasarlanan Cas sistemleri entegre hedef HIV-1 DNA&#39;sı 19.

<table><tbody><tr><td>DMEM HyClone</td><td>GE Healthcare</td><td>SH30243.01</td><td></td></tr><tr><td>FBS HyClone</td><td>GE Healthcare</td><td>SH30396.03</td><td></td></tr><tr><td>Penicillin/Streptomycin Gibco</td><td>Thermo Fisher</td><td>15140-122</td><td></td></tr><tr><td>Cell culture plates, 96 well, 24 well, 6 well.</td><td>Corning</td><td>353075, 353047, 353043</td><td></td></tr><tr><td>Micro tubes Axygen</td><td>Corning</td><td>311-08-051</td><td></td></tr><tr><td>Low molecular weight Poly I:C</td><td>InvivoGen</td><td>3182-29-6</td><td></td></tr><tr><td>DharmaFECT-1</td><td>Dharmacon</td><td>T-2001-01</td><td>transfection reagent for synthetic RNAs</td></tr><tr><td>TransIT-LT1</td><td>Mirus</td><td>MIR 2300</td><td>transfection reagent for RNA expression plasmids</td></tr><tr><td>Nonidet P40 (NP-40)</td><td>USB</td><td>19628</td><td></td></tr><tr><td>[&lt;sup&gt;32&lt;/sup&gt;P]dTTP</td><td>Perkin Elmer</td><td>BLU505H</td><td></td></tr><tr><td>poly(A) RNA template&amp;nbsp;</td><td>Sigma-Aldrich</td><td>10108626001</td><td></td></tr><tr><td>oligo(dT)&lt;sub&gt;12-18&lt;/sub&gt; DNA primer</td><td>Thermo Fisher</td><td>18418-012</td><td></td></tr><tr><td>DEAE filtermat paper&amp;nbsp;</td><td>Perkin Elmer</td><td>1450-522</td><td></td></tr><tr><td>Microplate scintillation counter</td><td>Perkin Elmer</td><td>1450-024</td><td></td></tr><tr><td>MTT</td><td>Sigma-Aldrich</td><td>M-2128</td><td></td></tr><tr><td>DPBS HyClone</td><td>GE Healthcare</td><td>SH30028.02</td><td></td></tr><tr><td>Microplate spectrophotometer</td><td>Bio-rad</td><td>1706930</td><td></td></tr><tr><td>Lysis buffer tablets</td><td>Roche</td><td>4693159001, 4906837001</td><td>protease and phosphatase inhibitors</td></tr><tr><td>Microcentrifuge</td><td>Eppendorf</td><td>5415R</td><td></td></tr><tr><td>Bradford reagent</td><td>Bio-rad</td><td>500-0006</td><td></td></tr><tr><td>Gel running chamber</td><td>Hoefer</td><td>SE600</td><td></td></tr><tr><td>Semi-dry transfer cell</td><td>Bio-rad</td><td>1703940</td><td></td></tr><tr><td>Protein ladder EZ-Run</td><td>Thermo Fisher</td><td>BP3603-500</td><td></td></tr><tr><td>Nitrocellulose membrane</td><td>Bio-rad</td><td>162-0094</td><td></td></tr><tr><td>BSA</td><td>Sigma-Aldrich</td><td>A9647-1006</td><td></td></tr><tr><td>Antibody stripping solution</td><td>Millipore</td><td>2504</td><td></td></tr><tr><td>ECL - Pierce</td><td>Thermo Fisher&amp;nbsp;</td><td>PI32106</td><td></td></tr><tr><td>ADAR1 antibody</td><td></td><td></td><td>from Dr. B.L. Bass</td></tr><tr><td>phospho-T446-PKR antibody</td><td>Abcam</td><td>ab32036</td><td></td></tr><tr><td>phospho-S396-IRF3 antibody</td><td>Cell Signaling</td><td>4947</td><td></td></tr><tr><td>PKR antibody</td><td></td><td></td><td>from Dr. A. Hovanessian</td></tr><tr><td>IRF3 antibody</td><td>Cell Signaling</td><td>11904</td><td></td></tr><tr><td>Actin antibody</td><td>Millipore</td><td>MAB1501</td><td></td></tr><tr><td>Peroxidase-labeled goat anti-rabbit</td><td>KPL</td><td>474-1506</td><td></td></tr><tr><td>Peroxidase-labeled goat anti-mouse</td><td>KPL</td><td>474-1806</td><td></td></tr><tr><td>Ponceau S&amp;nbsp;</td><td>Sigma-Aldrich</td><td>6226-79-5</td><td></td></tr></tbody></table>

evaluation of the efficacy and toxicity of rnas targeting hiv-1 production for use in gene or drug therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Prosedürleri genel şematik bir üç test RNA ve Şekil 2B&#39;de sağlanan bir kontrol RNA için bir örnek transfeksiyon planı, Şekil 2 &#39;de gösterilmiştir. Viral üretimi ve hücre yaşayabilirliği tahlilleri için, her bir test yapı için Okuması bir negatif kontrol normalize edilir. Her test RNA bitişiğindeki negatif kontrol normalize böylece çoğaltır, setler halinde transfekte edilir. Bu durum, örneğin, negatif kontroller tüm bi...

Watch this Scientific Journal Video about Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy at JoVE.com

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

Bir gen ya da ilaç terapisinde kullanım için, HIV-1 üretim yapan RNA&#39;lar etkinlik ve toksisite Değerlendirilmesi

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

evaluation-efficacy-toxicity-rnas-targeting-hiv-1-production-for-use

Research

JoVE Journal

Immunology and Infection

7.8K Görüntüleme.  Lady Davis Institute for Medical Research. Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones. 

Video: Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Methods Development for Blood Borne Macrophage Carriage of Nanoformulated Antiretroviral Drugs

Nanoformulated drugs can improve pharmacodynamics and bioavailability while serving also to reduce drug toxicities for antiretroviral (ART) medicines. To this end, our laboratory has applied the principles of nanomedicine to simplify ART regimens and as such reduce toxicities while improving compliance and drug pharmacokinetics. Simple and reliable methods for manufacturing nanoformulated ART (nanoART) are shown. Particles of pure drug are encapsulated by a thin layer of surfactant lipid coating and produced by fractionating larger drug crystals into smaller ones by either wet milling or high-pressure homogenization. In an alternative method free drug is suspended in a droplet of a polymer. Herein, drug is dissolved within a polymer then agitated by ultrasonication until individual nanosized droplets are formed. Dynamic light scattering and microscopic examination characterize the physical properties of the particles (particle size, charge and shape). Their biologic properties (cell uptake and retention, cytotoxicity and antiretroviral efficacy) are determined with human monocyte-derived macrophages (MDM). MDM are derived from human peripheral blood monocytes isolated from leukopacks using centrifugal elutriation for purification. Such blood-borne macrophages may be used as cellular transporters for nanoART distribution to human immunodeficiency virus (HIV) infected organs. We posit that the repackaging of clinically available antiretroviral medications into nanoparticles for HIV-1 treatments may improve compliance and positively affect disease outcomes.

Nanoparticles of indinavir, ritonavir, efavirenz and atazanavir were manufactured using wet milling, homogenization and ultrasonication. These nanoformulations, collectively termed nanoformulated antiretroviral therapy (nanoART), assessed macrophage-based drug delivery. Monocyte-derived macrophage nanoART uptake, retention and sustained release were determined. These preliminary studies suggest the potential of nanoART for clinical use.

Kan Bazlı Makrofaj Taşıma Nanoformulated antiretroviral ilaçlar Geliştirme Yöntemleri

Nanoformulated drugs can improve pharmacodynamics and bioavailability while serving also to reduce drug toxicities for antiretroviral (ART) medicines. To ...

Biyomühendislik

Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact&#160;virus replication. Here we present the application of this technology to uncovering host targets that specifically modulate the replication of Maraba virus, an oncolytic rhabdovirus, and vaccinia virus with the goal of enhancing therapy. While the protocol has been tested for use with oncolytic Maraba virus and oncolytic vaccinia virus, this approach is applicable to other oncolytic viruses and can also be utilized for identifying host targets that modulate virus replication in mammalian cells in general. This protocol describes the development and validation of an assay for high-throughput RNAi screening in mammalian cells, the key considerations and preparation steps important for conducting a primary high-throughput RNAi screen, and a step-by-step guide for conducting a primary high-throughput RNAi screen; in addition, it broadly outlines the methods for conducting secondary screen validation and tertiary validation studies. The benefit of high-throughput RNAi screening is that it allows one to catalogue, in an extensive and unbiased fashion, host factors that modulate any aspect of virus replication for which one can develop an in vitro assay such as infectivity, burst size, and cytotoxicity. It has the power to uncover biotherapeutic targets unforeseen based on current knowledge.

Here we describe a protocol for employing high-throughput RNAi screening to uncover host targets that can be manipulated to enhance oncolytic virus therapy, specifically rhabodvirus and vaccinia virus therapy, but it can be readily adapted to other oncolytic virus platforms or for discovering host genes that modulate virus replication generally.

Tedavi Oncolytic virusu modüle ana faktörleri tanımlamak için genom çapında RNAi tarama

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact&#160;virus ...

Kanser Araştırmaları

Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering (si)RNAs to inhibit the expression of complementary RNA transcripts is being exploited as a new class of therapeutics for a variety of diseases including HIV. Many previous reports have shown that novel RNAi-based anti-HIV/AIDS therapeutic strategies have considerable promise; however, a key obstacle to the successful therapeutic application and clinical translation of siRNAs is efficient delivery. Particularly, considering the safety and efficacy of RNAi-based therapeutics, it is highly desirable to develop a targeted intracellular siRNA delivery approach to specific cell populations or tissues. The HIV-1 gp120 protein, a glycoprotein envelope on the surface of HIV-1, plays an important role in viral entry into CD4 cells. The interaction of gp120 and CD4 that triggers HIV-1 entry and initiates cell fusion has been validated as a clinically relevant anti-viral strategy for drug discovery. 
Herein, we firstly discuss the selection and identification of 2'-F modified anti-HIV gp120 RNA aptamers. Using a conventional nitrocellulose filter SELEX method, several new aptamers with nanomolar affinity were isolated from a 50 random nt RNA library. In order to successfully obtain bound species with higher affinity, the selection stringency is carefully controlled by adjusting the conditions. The selected aptamers can specifically bind and be rapidly internalized into cells expressing the HIV-1 envelope protein. Additionally, the aptamers alone can neutralize HIV-1 infectivity. Based upon the best aptamer A-1, we also create a novel dual inhibitory function anti-gp120 aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. Further, we utilize the gp120 aptamer-siRNA chimeras for cell-type specific delivery of the siRNA into HIV-1 infected cells. This dual function chimera shows considerable potential for combining various nucleic acid therapeutic agents (aptamer and siRNA) in suppressing HIV-1 infection, making the aptamer-siRNA chimeras attractive therapeutic candidates for patients failing highly active antiretroviral therapy (HAART).

Several 2&#x2019;-Fluoro RNA aptamers against HIV-1Ba-L gp120 with nanomole affinity are isolated from a RNA library by in vitro SELEX procedure. A new dual inhibitory function anti-gp120 aptamer-siRNA chimera is created and shows considerable promise for systemic anti-HIV therapy.

Hücre tipi siRNA teslimat için özel anti-HIV gp120 aptamers Geliştirme

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering ...

İmmünoloji ve Enfeksiyon

DNA Vector-based RNA Interference to Study Gene Function in Cancer

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA (siRNA) in gene silencing1, RNAi has become a powerful research tool in gene function studies. Compared to genetic deletion, RNAi-mediated gene silencing possesses many advantages, such as the ease with which it is carried out and its suitability to most cell lines. Multiple studies have demonstrated the applications of RNAi technology in cancer research. In particular, the development of the DNA vector-based technology to produce small hairpin RNA (shRNA) driven by the U6 or H1 promoter has made long term and inducible gene silencing possible2,3. Its use in combination with genetically engineered viral vectors, such as lentivirus, facilitates high efficiencies of shRNA delivery and/or integration into genomic DNA for stable shRNA expression. 
We describe a detailed procedure using the DNA vector-based RNAi technology to determine gene function, including construction of lentiviral vectors expressing shRNA, lentivirus production and cell infection, and functional studies using a mouse xenograft model. 
Various strategies have been reported in generating shRNA constructs. The protocol described here employing PCR amplification and a 3-fragment ligation can be used to directly and efficiently generate shRNA-containing lentiviral constructs without leaving any extra nucleotide adjacent to a shRNA coding sequence. Since the shRNA-expression cassettes created by this strategy can be cut out by restriction enzymes, they can be easily moved to other vectors with different fluorescent or antibiotic markers. Most commercial transfection reagents can be used in lentivirus production. However, in this report, we provide an economic method using calcium phosphate precipitation that can achieve over 90% transfection efficiency in 293T cells. Compared to constitutive shRNA expression vectors, an inducible shRNA system is particularly suitable to knocking down a gene essential to cell proliferation. We demonstrate the gene silencing of Yin Yang 1 (YY1), a potential oncogene in breast cancer4,5, by a Tet-On inducible shRNA system and its effects on tumor formation. Research using lentivirus requires review and approval of a biosafety protocol by the Biosafety Committee of a researcher's institution. Research using animal models requires review and approval of an animal protocol by the Animal Care and Use Committee (ACUC) of a researcher's institution.

RNA interference (RNAi) possesses many advantages over gene knockout and has been broadly used as a tool in gene functional studies. The invention of DNA vector-based RNAi technology has made long term and inducible gene knockdown possible, and also increased the feasibility of gene silencing in vivo.

Kanser gen Fonksiyon Eğitim için DNA vektör tabanlı RNA Girişim

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA ...

Tıp

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

RNA Virüslerinin geniş spektrumlu Kimyasal Önleyicileri için yüksek verimli tarama

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.
The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.
This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.

Here we describe cellular cytotoxicity and single round infectivity assays that allow for the rapid and accurate screening of compounds to determine their cellular cytotoxicity (CC50) and IC50 values against WT and drug resistant HIV-1.

HIV ters transkriptaz ve integraz inhibitörleri hızlı taranması

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify ...

<meta charset="utf8"></head><body>Etkili ve Güvenli Karaciğer Geni Yıkımı için İntrasplenik AAV8-shRNA Enjeksiyonu

Efficient Gene Knockdown in the Liver via Intrasplenic Injection of Adeno-Associated Virus Serotype 8 (AAV8)-Delivered Small Hairpin RNA

The liver is a major organ that performs essential metabolic functions. Developing an efficient and safe method to knock down gene expression in the liver provides an important tool for determining gene function in liver pathophysiology. In this study, we describe a method for intrasplenic injection of adeno-associated virus serotype 8 (AAV8) engineered to express a small hairpin RNA (shRNA) against a target gene of interest, nucleostemin (NS). Intrasplenic injection of AAV8 expressing an NS-targeting shRNA (AAV8-shNS1) achieved the same knockdown efficiency of NS in the liver as did portal vein injection, compared to the injection of AAV8 expressing a scrambled sequence shRNA (AAV8-shScr). Furthermore, injection of the AAV8-shRNA virus triggered minimal inflammatory reactions in the liver parenchyma. Most importantly, this intrasplenic injection protocol was not technically demanding and caused minimal bleeding at the injection site, which is the leading cause of perioperative and postoperative mortality when performing portal vein injection. This study reports an improved and relatively safe method to achieve efficient gene knockdown in the liver.

<meta charset="utf8"></head><body>Adeno-ilişkili virüs serotip 8 (AAV8) ile verilen küçük firkete RNA'sının intrasplenik enjeksiyonu yoluyla karaciğerde etkili gen yıkımı

This protocol describes how intrasplenic injection of AAV8-delivered small hairpin RNA achieves the same gene knockdown efficiency in the liver as portal vein injection, representing a simpler procedure with much lower perioperative and postoperative mortality and complications.

Adeno-ilişkili virüs serotip 8 (AAV8) ile verilen küçük firkete RNA'sının intrasplenik enjeksiyonu yoluyla karaciğerde etkili gen yıkımı

The liver is a major organ that performs essential metabolic functions. Developing an efficient and safe method to knock down gene expression in the liver ...

Quantitative Structure-Activity Relationship, Activity Prediction, and Molecular Dynamics of Non-nucleotide Reverse Transcriptase Inhibitors

The increasing incidence of HIV-1 drug resistance presents a challenge to the effectiveness of combination antiretroviral therapy, particularly in Southern Africa. The development of resistance to non-nucleotide reverse transcriptase inhibitors (NNRTIs) threatens the long-term success of antiretroviral therapy. In 2019, antimicrobial resistance directly accounted for an estimated 1.27 million deaths globally. This study employed an in-silico approach to investigate NNTRI drugs and their derivatives. Techniques used included density functional theory calculations, molecular docking, enumeration, quantitative structure-activity relationship (QSAR) analysis, molecular dynamics simulation (MDS), and molecular mechanics with generalized Born and surface area methods. The analysis focused on various pyrimidine derivatives and six NNRTI drugs, examining their interactions with the HIV-1 protein (PDB code 1HQU).
A QSAR model was developed to predict the biological activity of the six NNRTIs under investigation. Using 94 pyrimidine derivatives, the QSAR model achieved an R2 OF 0.822 and a Q2 of 0.815, indicating a high level of predictive accuracy.
MDS were conducted to assess the stability of various ligands and their newly developed alternatives, ensuring they remained bound to the protein&#39;s active site over a 200-nanosecond simulation timeframe. Etravirine exhibited root mean square deviation (RMSD) fluctuations of approximately 4.5 &#197;, while its enumerated derivatives showed RMSD fluctuations of 3.5 &#197;. Through molecular docking, MDS, and free energy computations, enumerated Etravirine demonstrated the best performance, with an activity value of 7.373 and a docking score of -10.517 kcal/mol. Furthermore, the calculated free energy of binding for enumerated Etravirine was -89.684 kcal/mol, outperforming other ligands under investigation. The significant improvement suggests that the modified Etravirine holds promising potential as a novel agent in antiretroviral therapy.
The obtained lower RMSD value, the enhanced amino acid interactions, and the highest binding free energy indicate that enumerated Etravirine could serve as a viable alternative for HIV/AIDS treatment.

This study used in-silico strategies to identify Enumerated Etravirine as a promising therapeutic agent for HIV. Our findings on molecular interactions and dynamics support the rational design of novel NNRTIs as possible HIV treatment alternatives.

Bir gen ya da ilaç terapisinde kullanım için, HIV-1 üretim yapan RNA'lar etkinlik ve toksisite Değerlendirilmesi

Özet

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Nükleotid Olmayan Ters Transkriptaz İnhibitörlerinin Kantitatif Yapı-Aktivite İlişkisi, Aktivite Tahmini ve Moleküler Dinamiği