Name: evaluation of the efficacy and toxicity of rnas targeting hiv-1 production for use in gene or drug therapy
Uploaded: 2016-09-05T14:00:00
Description: 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

여기에 제시된 작품은 건강 연구 (CIHR)의 캐나다 연구소에 의해 지원되었다 (DCB-120266, PPP-133377와 AG에 HBF-348967을 부여).

1. 세포와 형질 <ol><li> 둘 베코 변성 이글 배지 (DMEM)에서 배양 HEK 293T 세포를 10 % 소 태아 혈청 (FBS) 및 1 % 페니실린 / 스트렙토 마이신이 보충. 세포 배양 배지에서 2 × 105 세포 / ㎖ 현탁액을 준비한다. 의 각 웰에 세포 현탁액의 500, 100 및 1,000 μl를 추가, 24도, 96도, 12 웰 플레이트, 바이러스 생산, 세포 생존 및 면역 활성 ​​분석, 각각 (그림 2A)합니다. </li><li> 부드럽게 플레이?...

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	<li>Hoxie, J. A., June, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+cell+and+gene+therapies+for+HIV.">Novel cell and gene therapies for HIV.</a> Cold Spring Harb Perspect Med. 2, a007179 (2012).</li><li>Bobbin, M. L., Burnett, J. C., Rossi, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference+approaches+for+treatment+of+HIV-1+infection.">RNA interference approaches for treatment of HIV-1 infection.</a> Genome Med. 7, 50 (2015).</li><li>Allers, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+the+cure+of+HIV+infection+by+CCR5Delta32/Delta32+stem+cell+transplantation.">Evidence for the cure of HIV infection by CCR5Delta32/Delta32 stem cell transplantation.</a> Blood. 117, 2791-2799 (2011).</li><li>DiGiusto, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+hematopoietic+stem+cell+based+gene+therapy+for+HIV-1+infection:+considerations+for+proof+of+concept+studies+and+translation+to+standard+medical+practice.">Development of hematopoietic stem cell based gene therapy for HIV-1 infection: considerations for proof of concept studies and translation to standard medical practice.</a> Viruses. 5, 2898-2919 (2013).</li><li>Burke, B. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+Cellular+Resistance+to+HIV-1+Infection+In+Vivo+Using+a+Dual+Therapeutic+Lentiviral+Vector.">Engineering Cellular Resistance to HIV-1 Infection In Vivo Using a Dual Therapeutic Lentiviral Vector.</a> Mol Ther Nucleic Acids. 4, e236 (2015).</li><li>DiGiusto, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-based+gene+therapy+for+HIV+with+lentiviral+vector-modified+CD34(+)+cells+in+patients+undergoing+transplantation+for+AIDS-related+lymphoma.">RNA-based gene therapy for HIV with lentiviral vector-modified CD34(+) cells in patients undergoing transplantation for AIDS-related lymphoma.</a> Sci Transl Med. 2, 36ra43 (2010).</li><li>Scarborough, R. J., Gatignol, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV+and+Ribozymes.">HIV and Ribozymes.</a> Adv Exp Med Biol. 848, 97-116 (2015).</li><li>Eekels, J. J., Berkhout, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+a+durable+treatment+of+HIV-1+infection+using+RNA+interference.">Toward a durable treatment of HIV-1 infection using RNA interference.</a> Prog Mol Biol Transl Sci. 102, 141-163 (2011).</li><li>Blazquez, L., Fortes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=U1+interference+(U1i)+for+Antiviral+Approaches.">U1 interference (U1i) for Antiviral Approaches.</a> Adv Exp Med Biol. 848, 51-69 (2015).</li><li>McIntyre, G. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=96+shRNAs+designed+for+maximal+coverage+of+HIV-1+variants.">96 shRNAs designed for maximal coverage of HIV-1 variants.</a> Retrovirology. 6, 55 (2009).</li><li>Sajic, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+modified+U1+snRNAs+to+inhibit+HIV-1+replication.">Use of modified U1 snRNAs to inhibit HIV-1 replication.</a> Nucleic Acids Res. 35, 247-255 (2007).</li><li>Low, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SHAPE-directed+discovery+of+potent+shRNA+inhibitors+of+HIV-1.">SHAPE-directed discovery of potent shRNA inhibitors of HIV-1.</a> Mol Ther. 20, 820-828 (2012).</li><li>Scarborough, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Conserved+Target+Site+in+HIV-1+Gag+RNA+is+Accessible+to+Inhibition+by+Both+an+HDV+Ribozyme+and+a+Short+Hairpin+RNA.">A Conserved Target Site in HIV-1 Gag RNA is Accessible to Inhibition by Both an HDV Ribozyme and a Short Hairpin RNA.</a> Mol Ther Nucleic Acids. 3, e178 (2014).</li><li>Lain&#233;, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+cleavage+of+HIV-1+RNA+by+new+SOFA-HDV+ribozymes+and+their+potential+to+inhibit+viral+replication.">In vitro and in vivo cleavage of HIV-1 RNA by new SOFA-HDV ribozymes and their potential to inhibit viral replication.</a> RNA Biol. 8, 343-353 (2011).</li><li>Scarborough, R. J., L&#233;vesque, M. V., Perreault, J. P., Gatignol, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+Evaluation+of+Clinically+Relevant+SOFA-HDV+Ribozymes+Targeting+HIV+RNA.">Design and Evaluation of Clinically Relevant SOFA-HDV Ribozymes Targeting HIV RNA.</a> Methods Mol Biol. 1103, 31-43 (2014).</li><li>Scarborough, R. J., Adams, K. L., Daher, A., Gatignol, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+Inhibition+of+HIV-1+Production+by+Short+Hairpin+RNAs+and+Small+Interfering+RNAs+Targeting+a+Highly+Conserved+Site+in+HIV-1+Gag+RNA+Is+Optimized+by+Evaluating+Alternative+Length+Formats.">Effective Inhibition of HIV-1 Production by Short Hairpin RNAs and Small Interfering RNAs Targeting a Highly Conserved Site in HIV-1 Gag RNA Is Optimized by Evaluating Alternative Length Formats.</a> Antimicrob Agents Chemother. 59, 5297-5305 (2015).</li><li>Sarzotti-Kelsoe, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+and+validation+of+the+TZM-bl+assay+for+standardized+assessments+of+neutralizing+antibodies+against+HIV-1.">Optimization and validation of the TZM-bl assay for standardized assessments of neutralizing antibodies against HIV-1.</a> J Immunol Methods. 409, 131-146 (2014).</li><li>Campos, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+lasting+control+of+viral+rebound+with+a+new+drug+ABX464+targeting+Rev+-+mediated+viral+RNA+biogenesis.">Long lasting control of viral rebound with a new drug ABX464 targeting Rev - mediated viral RNA biogenesis.</a> Retrovirology. 12, 1-15 (2015).</li><li>Zhu, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CRISPR/Cas9+system+inactivates+latent+HIV-1+proviral+DNA.">The CRISPR/Cas9 system inactivates latent HIV-1 proviral DNA.</a> Retrovirology. 12, 22 (2015).</li><li>Bounou, S., Leclerc, J. E., Tremblay, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presence+of+host+ICAM-1+in+laboratory+and+clinical+strains+of+human+immunodeficiency+virus+type+1+increases+virus+infectivity+and+CD4(+)-T-cell+depletion+in+human+lymphoid+tissue,+a+major+site+of+replication+in+vivo.">Presence of host ICAM-1 in laboratory and clinical strains of human immunodeficiency virus type 1 increases virus infectivity and CD4(+)-T-cell depletion in human lymphoid tissue, a major site of replication in vivo.</a> J Virol. 76, 1004-1014 (2002).</li><li>Shan, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+PCR+assay+for+quantification+of+HIV-1+RNA.">A novel PCR assay for quantification of HIV-1 RNA.</a> J Virol. 87, 6521-6525 (2013).</li><li>Clerzius, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+PKR+activator,+PACT,+becomes+a+PKR+inhibitor+during+HIV-1+replication.">The PKR activator, PACT, becomes a PKR inhibitor during HIV-1 replication.</a> Retrovirology. 10, 96 (2013).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal Biochem. 72, 248-254 (1976).</li><li>Stoscheck, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+protein.">Quantitation of protein.</a> Methods Enzymol. 182, 50-68 (1990).</li><li>Battisti, P. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Additive+activity+between+the+trans-activation+response+RNA-binding+protein,+TRBP2,+and+cyclin+T1+on+HIV+type+1+expression+and+viral+production+in+murine+cells.">Additive activity between the trans-activation response RNA-binding protein, TRBP2, and cyclin T1 on HIV type 1 expression and viral production in murine cells.</a> AIDS Res Hum Retroviruses. 19, 767-778 (2003).</li><li>Bannwarth, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-specific+regulation+of+TRBP1+promoter+by+NF-Y+transcription+factor+in+lymphocytes+and+astrocytes.">Cell-specific regulation of TRBP1 promoter by NF-Y transcription factor in lymphocytes and astrocytes.</a> J Mol Biol. 355, 898-910 (2006).</li><li>Clerzius, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ADAR1+interacts+with+PKR+during+human+immunodeficiency+virus+infection+of+lymphocytes+and+contributes+to+viral+replication.">ADAR1 interacts with PKR during human immunodeficiency virus infection of lymphocytes and contributes to viral replication.</a> J Virol. 83, 10119-10128 (2009).</li><li>Burugu, S., Daher, A., Meurs, E. F., Gatignol, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+translation+and+its+regulation+by+cellular+factors+PKR+and+PACT.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+of+HIV-1+with+RNA+interference:+a+multiple+shRNA+approach.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+design+and+validation+of+antiviral+siRNA+for+targeting+HIV-1.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excessive+RNA+splicing+and+inhibition+of+HIV-1+replication+induced+by+modified+U1+small+nuclear+RNAs.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+expression+and+HIV-1+inhibition+of+chimeric+tRNA(Lys3)-ribozymes+under+dual+U6+snRNA+and+tRNA+promoters.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentiviral+siRNAs+targeting+multiple+highly+conserved+RNA+sequences+of+human+immunodeficiency+virus+type+1.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+RRE+RNA+acts+as+an+RNA+silencing+suppressor+by+competing+with+TRBP-bound+siRNAs.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+delivery+of+small+interfering+RNAs+reduces+RNAi+efficacy+by+selective+incorporation+into+RISC.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=U1+adaptors+result+in+reduction+of+multiple+pre-mRNA+species+principally+by+sequestering+U1snRNP.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+Viral+Entry+Assays+for+the+Identification+and+Evaluation+of+Antiviral+Compounds.">Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds.</a> J Vis Exp. , (2015).</li><li>Riss, T. L., et al., Sittampalam, G. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Assay Guidance Manual. , (2004).</li><li>Reynolds, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+the+interferon+response+by+siRNA+is+cell+type-+and+duplex+length-dependent.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+or+stimulation?+siRNA+delivery+and+the+immune+system.">Silencing or stimulation? siRNA delivery and the immune system.</a> Annu Rev Chem Biomol Eng. 2, 77-96 (2011).</li></ol>

기재된 HIV-1 생산 분석법 HEK293T 셀들 (도 2)를 이용하여 수행하고, 효과적인 리보 자임 (13) shRNA를 10,29, siRNA를 30 및 U1i RNA 11,31 표적 부위를 HIV-1 RNA를 선별하는 데 사용되는 분석 비슷 하였다. HIV-1 생성을 정량화하기 위해 다양한 방법을 사용하여, 대부분의 연구는 후보의 RNA와 HIV-1 발현 플라스미드의 공동 형질 감염 후 바이러스 생산을 48 시간으로 측정...

현재 HIV-1 치료의 한계는 만성적 질환의 진행을 방지하기 위해 투여되어야한다는 것이다. HIV-1에 강한 T 림프구 또는 조혈 줄기 세포 이식, 약물 치료 -1,2-의 부재 HIV-1 복제의 장기간 제어를 제공하는 가능성을 가지며 또한 HIV-1 치료를 달성하기위한 효과적인 방법이 될 수있다 3. HIV-1 복제에 대해 내성 세포를 렌더링하기위한 한 가지 방법은자가 이식 동안 4 감염된 개인의 세포에 항 HIV-1 RNA를 또는 펩타이드를 코딩하는 하나 이상의 유전자를 삽입하는 것이다. 여러 후보 항 HIV-1 유전자는 단일 유전자에 HIV-1의 저항 현상을 방지하기 위해, 두 개 또는 세 개의 5 (6)의 조합에 일부 진입하여 임상 시험을 고안 하였다. 항 HIV-1 RNA를 인해 면역 반응을 유도하는 그들의 낮은 전위로하고 있기 때문에 결합 유전자 치료에 대한 상위 후보자들이다매우 짧은 유전자 서열로부터 전사된다. 일부 항 HIV-1 RNA를이 바이러스 항목 및 통합을 목표로 설계되었습니다. 그러나, 대부분의 항 HIV-1 RNA를는 바이러스 라이프 사이클 후 통합 단계 (도 1)을 대상. 후 통합 억제제는 리보 자임 (7), shRNA를 8 U1i RNA를 9로는 HIV-1 조절 단백질 문신 또는 계 1, 및 안티센스 기반의 RNA를 대상으로 HIV-1 RNA에 다른 사이트를 대상으로, 미끼 RNA를 포함. 항 HIV-1 RNA를의 효능을 비교하기 위해 사용 된 방법은 유전자 후보 RNA를 코딩하는 바이러스 성 일시적 후보의 RNA를 발현하는 플라스미드로 형질 감염된 세포에서 생산 및 HIV-1 발현 플라스미드를 측정 형질 세포에서 바이러스 복제를 모니터링 포함 10 -13. 우리는 이전에 새로운 리보 자임 대상 사이트 13 ~ 15에 대한 HIV-1 RNA를 선별하는 HIV-1 생산 분석을 사용했다. 이러한 방법은 RNA 인 이후의 포맷을 최적화하기 위해 정제 된간섭은 분자의 shRNA로 플라스미드 DNA로부터 발현 또는 합성 siRNA를 16로 전달. 상기 분석은 인간 배아 신장 (HEK) 293T 세포로부터 성숙 바이러스의 생산을 측정하고 (도 1) HIV-1 복제 사이클 후 통합 단계를 타겟팅 억제제의 효과를 비교하기 위해 사용될 수있다. 사전 통합 단계를 타겟팅 억제제를 들면, TZM-BL 세포 감염 분석 (17)와 같은 다른 분석은 항 바이러스 효능을 평가하기 위해 필요하다. 병원에서 항 HIV-1 RNA를의 전달을위한 주요 안전 문제는 인간의 RNA 또는 단백질과 선천성 면역 센서의 작동에 잠재적 인 오프 대상 효과를 포함한다. 항 HIV-1 siRNA의 독성을 평가하기 위해 다른 세포주 16 세포 생존력 분석을 이용했다. 우리는 또한 이중 가닥 RNA 면역 센서의 활성화를 측정 RNA 활성화 단백질 키나제 R (PKR) 및 수용체 3 (TLR3) 등 수신자뿐 아니라 전인터페론의 Pression의 유전자, ADAR1의 P150을 자극. 이러한 분석은 항 HIV-1 RNA를 효능은 세포 생존 또는 면역 센서의 활성화에 대한 간접적 인 효과에 기인 아니라는 것을 확인하는데 사용될 수있다. 또한 발전에서 잠재적 인 독성과 후보 RNA를 제외 유용하다. 다음 프로토콜에서, 절차는 새로운 치료의 RNA를 확인하고, 기존의 형식을 설명하는 최적화. 방법은 HIV-1 복제의 심사 RNA 기반 후 통합 억제제에 유용과 같은 바이러스 성 RNA 18 CRISPR의 레브 매개 수출을 대상으로 작은 분자와 같은 다른 후 통합 억제제를 화면에 적용 할 수 / 설계 카스 시스템은 통합 대상으로 HIV-1 DNA 19.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>[32P]dTTP</td>
			<td>Perkin Elmer</td>
			<td>BLU505H</td>
			<td></td>
		</tr>
		<tr>
			<td>poly(A) RNA template&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>10108626001</td>
			<td></td>
		</tr>
		<tr>
			<td>oligo(dT)12-18 DNA primer</td>
			<td>Thermo Fisher</td>
			<td>18418-012</td>
			<td></td>
		</tr>
		<tr>
			<td>DEAE filtermat paper&#160;</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&#160;</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&#160;</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.

절차의 일반적인 개략도 세 시험 RNA를도 2b에 제공되는 제어 RNA위한 예시 형질 계획도 2에 도시되어있다. 바이러스 생산 및 세포 생존 분석을 위해 각 테스트 구조에 대한 판독은 음성 대조군으로 정규화된다. 각 시험 RNA가 인접 대조군에 정상화되도록 복제가 세트로 형질 감염된다. 이것은 예를 들어, 음성 대조군 모두 제 형질, 경우 발생할 수 착...

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.

유전자 또는 약물 치료에 사용하기 위해 HIV-1 생산을 표적 RNA를의 효능 및 독성 평가

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 ...

Research

JoVE Journal

Immunology and Infection

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

V3의 HIV - 1 Tropism의 Genotypic 추론 사용 인구를 기반 시퀀싱

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

연구

면역학 및 감염병학

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

준비 및 사용 HIV - 1 차 감염된 CD4 + 대상 세포로 T 세포 자연 킬러 세포 세포 독성 Assays에서

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Multivirus 특정 T 세포 생성 Allogeneic 조혈 줄기 세포 이식 후 바이러스 감염을 예방 / 치료

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays

표준 임상 Assays에 의해 탐지의 한도 이하 바이러스성로드와 HIV 감염된 개인의 확장 및 HIV - 1 RNA를 Quantifying

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below ...

In vitro Uncoating of HIV-1 Cores

In vitro Uncoating of HIV-1 Cores

HIV - 1 코어의 시험 관내 Uncoating에서

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is ...

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

의 이미징 HIV-1 봉투 유발 Virological 시냅스와 합성 지질 Bilayers에 신호

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

셀 기반의 ELISA 분석을 사용하여 HIV-1 삼량 봉투 당 단백질의 구조적 평가

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

높은 처리량 정량 실시간 RT-PCR 분석 유형 I과 III 인터페론 하위 유형의 발현 양상을 결정하는

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

HIV-1 양성 개인의 플라즈마에서 엑소 좀의 분리

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. ...

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

측정<em&gt; 체외</em&gt; HIV-1 사전 통합 단지의 통합 활동

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the ...