Synthetic gRNA/Cas9 Ribonucleoprotein Inhibits HIV Reactivation and Replication.
Synthetic gRNA/Cas9 Ribonucleoprotein Inhibits HIV Reactivation and Replication.
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DOI：
10.3390/v14091902
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发表时间：
2022-08-28
期刊：
影响因子：
--
通讯作者：
Yao ZQ
中科院分区：
文献类型：
--
作者：
Khanal S;Cao D;Zhang J;Zhang Y;Schank M;Dang X;Nguyen LNT;Wu XY;Jiang Y;Ning S;Zhao J;Wang L;Gazzar ME;Moorman JP;Yao ZQ
目前针对人类免疫缺陷病毒（HIV）的抗逆转录病毒疗法（ART）可以阻止病毒复制，但不能根除HIV感染，因为整合到宿主基因组中的原病毒DNA在储存库细胞中保持基因沉默，并且在ART中断或停止时具有复制能力。基于CRISPR/ cas9的技术被广泛用于通过诱变（即核苷酸插入/删除和/或替代）编辑靶基因，从而可以灭活整合的原病毒DNA。然而，CRISPR/Cas9传递系统通常需要病毒载体，这给人类治疗应用带来了安全问题。在这项研究中，我们使用合成的指导RNA (gRNA)/ cas9核糖核蛋白（RNP）作为非病毒制剂来开发一种新的HIV基因疗法。我们设计了一系列针对不同HIV复制关键基因的grna，并在淋巴细胞和单核细胞潜伏HIV细胞系中测试了它们的抗病毒效果和细胞毒性。与scramble gRNA对照相比，HIV-gRNA/Cas9 rnp处理的细胞表现出有效的病毒抑制，没有明显的细胞毒性，这可以通过显著抑制潜伏的HIV DNA再激活和RNA复制来证明。此外，HIV- grna /Cas9 RNP抑制p24抗原的表达，抑制感染性病毒颗粒的产生，并在靶向HIV基因中产生特异性的DNA切割，DNA测序证实了这一点。由于其快速的DNA切割、低脱靶效应、低插入突变风险、易于生产和准备用于临床应用，本研究提供了一个概念证明，合成gRNA/Cas9 RNP药物可以作为一种新的治疗方法用于根除HIV。
The current antiretroviral therapy (ART) for human immunodeficiency virus (HIV) can halt viral replication but cannot eradicate HIV infection because proviral DNA integrated into the host genome remains genetically silent in reservoir cells and is replication-competent upon interruption or cessation of ART. CRISPR/Cas9-based technology is widely used to edit target genes via mutagenesis (i.e., nucleotide insertion/deletion and/or substitution) and thus can inactivate integrated proviral DNA. However, CRISPR/Cas9 delivery systems often require viral vectors, which pose safety concerns for therapeutic applications in humans. In this study, we used synthetic guide RNA (gRNA)/Cas9-ribonucleoprotein (RNP) as a non-viral formulation to develop a novel HIV gene therapy. We designed a series of gRNAs targeting different HIV genes crucial for HIV replication and tested their antiviral efficacy and cellular cytotoxicity in lymphoid and monocytic latent HIV cell lines. Compared with the scramble gRNA control, HIV-gRNA/Cas9 RNP-treated cells exhibited efficient viral suppression with no apparent cytotoxicity, as evidenced by the significant inhibition of latent HIV DNA reactivation and RNA replication. Moreover, HIV-gRNA/Cas9 RNP inhibited p24 antigen expression, suppressed infectious viral particle production, and generated specific DNA cleavages in the targeted HIV genes that are confirmed by DNA sequencing. Because of its rapid DNA cleavage, low off-target effects, low risk of insertional mutagenesis, easy production, and readiness for use in clinical application, this study provides a proof-of-concept that synthetic gRNA/Cas9 RNP drugs can be utilized as a novel therapeutic approach for HIV eradication.
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影响因子：
3.7
作者：
Altmann T;Gennery AR
通讯作者：
Gennery AR
DOI：
10.1016/j.omtn.2018.05.021
发表时间：
2018-09-07
期刊：
Molecular therapy. Nucleic acids
影响因子：
--
作者：
Bella R;Kaminski R;Mancuso P;Young WB;Chen C;Sariyer R;Fischer T;Amini S;Ferrante P;Jacobson JM;Kashanchi F;Khalili K
通讯作者：
Khalili K
影响因子：
46.9
作者：
Doench JG;Fusi N;Sullender M;Hegde M;Vaimberg EW;Donovan KF;Smith I;Tothova Z;Wilen C;Orchard R;Virgin HW;Listgarten J;Root DE
通讯作者：
Root DE
影响因子：
--
作者：
Atkins A;Chung CH;Allen AG;Dampier W;Gurrola TE;Sariyer IK;Nonnemacher MR;Wigdahl B
通讯作者：
Wigdahl B
CRISPR-CAS9介导的基因失活的高度活性SGRNA的合理设计。
影响因子：
46.9
作者：
Doench, John G.;Hartenian, Ella;Graham, Daniel B.;Tothova, Zuzana;Hegde, Mudra;Smith, Ian;Sullender, Meagan;Ebert, Benjamin L.;Xavier, Ramnik J.;Root, David E.
通讯作者：
Root, David E.