Abstract:Transcription activator-like effector nuclease (TALEN) represents a valuable tool for genomic engineering due to its single-nucleotide precision, high nuclease activity, and low cytotoxicity. We report here systematic design and characterization of 28 novel TALENs targeting multiple regions of CCR5 gene (CCR5-TALEN) which encodes the co-receptor critical for entry of human immunodeficiency virus type I (HIV-1). By systemic characterization of these CCR5-TALENs, we have identified one (CCR5-TALEN-515) with high… Show more
“…From previous studies, HIV-1 enters cells via co-receptor CCR5 at the early stage of infection and many studies involving CCR5 disruption have been performed by utilizing gene manipulation tools, such as ZFN, TALEN, and the newly developed CRISPR/Cas9 [ 22 , 33 , 43 , 44 ]. Meanwhile, CXCR4, which may act as a co-receptor for late stage entry and accelerate the progress of the disease [ 34 ], has also been studied [ 35 , 37 ].…”
BackgroundThe main approach to treat HIV-1 infection is combination antiretroviral therapy (cART). Although cART is effective in reducing HIV-1 viral load and controlling disease progression, it has many side effects, and is expensive for HIV-1 infected patients who must remain on lifetime treatment. HIV-1 gene therapy has drawn much attention as studies of genome editing tools have progressed. For example, zinc finger nucleases (ZFN), transcription activator like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 have been utilized to successfully disrupt the HIV-1 co-receptors CCR5 or CXCR4, thereby restricting HIV-1 infection. However, the effects of simultaneous genome editing of CXCR4 and CCR5 by CRISPR-Cas9 in blocking HIV-1 infection in primary CD4+ T cells has been rarely reported. Furthermore, combination of different target sites of CXCR4 and CCR5 for disruption also need investigation.ResultsIn this report, we designed two different gRNA combinations targeting both CXCR4 and CCR5, in a single vector. The CRISPR-sgRNAs-Cas9 could successfully induce editing of CXCR4 and CCR5 genes in various cell lines and primary CD4+ T cells. Using HIV-1 challenge assays, we demonstrated that CXCR4-tropic or CCR5-tropic HIV-1 infections were significantly reduced in CXCR4- and CCR5-modified cells, and the modified cells exhibited a selective advantage over unmodified cells during HIV-1 infection. The off-target analysis showed that no non-specific editing was identified in all predicted sites. In addition, apoptosis assays indicated that simultaneous disruption of CXCR4 and CCR5 in primary CD4+ T cells by CRISPR-Cas9 had no obvious cytotoxic effects on cell viability.ConclusionsOur results suggest that simultaneous genome editing of CXCR4 and CCR5 by CRISPR-Cas9 can potentially provide an effective and safe strategy towards a functional cure for HIV-1 infection.Electronic supplementary materialThe online version of this article (doi:10.1186/s13578-017-0174-2) contains supplementary material, which is available to authorized users.
“…From previous studies, HIV-1 enters cells via co-receptor CCR5 at the early stage of infection and many studies involving CCR5 disruption have been performed by utilizing gene manipulation tools, such as ZFN, TALEN, and the newly developed CRISPR/Cas9 [ 22 , 33 , 43 , 44 ]. Meanwhile, CXCR4, which may act as a co-receptor for late stage entry and accelerate the progress of the disease [ 34 ], has also been studied [ 35 , 37 ].…”
BackgroundThe main approach to treat HIV-1 infection is combination antiretroviral therapy (cART). Although cART is effective in reducing HIV-1 viral load and controlling disease progression, it has many side effects, and is expensive for HIV-1 infected patients who must remain on lifetime treatment. HIV-1 gene therapy has drawn much attention as studies of genome editing tools have progressed. For example, zinc finger nucleases (ZFN), transcription activator like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 have been utilized to successfully disrupt the HIV-1 co-receptors CCR5 or CXCR4, thereby restricting HIV-1 infection. However, the effects of simultaneous genome editing of CXCR4 and CCR5 by CRISPR-Cas9 in blocking HIV-1 infection in primary CD4+ T cells has been rarely reported. Furthermore, combination of different target sites of CXCR4 and CCR5 for disruption also need investigation.ResultsIn this report, we designed two different gRNA combinations targeting both CXCR4 and CCR5, in a single vector. The CRISPR-sgRNAs-Cas9 could successfully induce editing of CXCR4 and CCR5 genes in various cell lines and primary CD4+ T cells. Using HIV-1 challenge assays, we demonstrated that CXCR4-tropic or CCR5-tropic HIV-1 infections were significantly reduced in CXCR4- and CCR5-modified cells, and the modified cells exhibited a selective advantage over unmodified cells during HIV-1 infection. The off-target analysis showed that no non-specific editing was identified in all predicted sites. In addition, apoptosis assays indicated that simultaneous disruption of CXCR4 and CCR5 in primary CD4+ T cells by CRISPR-Cas9 had no obvious cytotoxic effects on cell viability.ConclusionsOur results suggest that simultaneous genome editing of CXCR4 and CCR5 by CRISPR-Cas9 can potentially provide an effective and safe strategy towards a functional cure for HIV-1 infection.Electronic supplementary materialThe online version of this article (doi:10.1186/s13578-017-0174-2) contains supplementary material, which is available to authorized users.
“…TALENs are also showing promise in other strategies. Like ZFPs, TALENs have been used to knock out the CCR5 gene in cell culture [203][204][205][206], permitting efficient gene editing and protection of cells from HIV infection [207] with minimal off-target activity and low cytotoxicity. TALE-activators, corresponding to TALEs fused to potent transcription activators, have been developed and shown to effectively reactivate latent HIV-1 in cell line models [208][209][210] and in PBMCs from ART-suppressed individuals [210].…”
Human immunodeficiency virus (HIV-1) indefinitely persists, despite effective antiretroviral therapy (ART), within a small pool of latently infected cells. These cells often display markers of immunologic memory and harbor both replication-competent and -incompetent proviruses at approximately a 1:100 ratio. Although complete HIV eradication is a highly desirable goal, this likely represents a bridge too far for our current and foreseeable technologies. A more tractable goal involves engineering a sustained viral remission in the absence of ART--a "functional cure." In this setting, HIV remains detectable during remission, but the size of the reservoir is small and the residual virus is effectively controlled by an engineered immune response or other intervention. Biological precedence for such an approach is found in the post-treatment controllers (PTCs), a rare group of HIV-infected individuals who, following ART withdrawal, do not experience viral rebound. PTCs are characterized by a small reservoir, greatly reduced inflammation, and the presence of a poorly understood immune response that limits viral rebound. Our goal is to devise a safe and effective means for replicating durable post-treatment control on a global scale. This requires devising methods to reduce the size of the reservoir and to control replication of this residual virus. In the following sections, we will review many of the approaches and tools that likely will be important for implementing such a "reduce and control" strategy and for achieving a PTC-like sustained HIV remission in the absence of ART.
“…It has been shown that the presence of multiple sequence repeats in TALEN genes renders them unsuitable cargos for lentiviral vector repeats [81]. However, the single-nucleotide precision give rise to superior editing efficiency with minimal off-target and cytotoxicity effects when compared to ZFNs thereby making TALENs good candidates for sequence-specific genome modification [82]. Fig.…”
“…However, several experimental studies have shown promising results with potential for optimization for large scale anti-HIV-1 treatment. Shi and colleagues recently generated 28 CCR5-TALEN screens to target several domains on the CCR5 of CD4+ T cells [82]. CCR5-ZFNs similar to the anti-HIV-1 ZFNs currently being applied for ongoing clinical trials were used as controls in this study for comparative studies.…”
Current treatment for HIV-1 largely relies on chemotherapy through the administration of antiretroviral drugs. While the search for anti-HIV-1 vaccine remain elusive, the use of highly active antiretroviral therapies (HAART) have been far-reaching and has changed HIV-1 into a manageable chronic infection. There is compelling evidence, including several side-effects of ARTs, suggesting that eradication of HIV-1 cannot depend solely on antiretrovirals. Gene therapy, an expanding treatment strategy, using RNA interference (RNAi) and programmable nucleases such as meganuclease, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins (CRISPR–Cas9) are transforming the therapeutic landscape of HIV-1. TALENS and ZFNS are structurally similar modular systems, which consist of a FokI endonuclease fused to custom-designed effector proteins but have been largely limited, particularly ZFNs, due to their complexity and cost of protein engineering. However, the newly developed CRISPR–Cas9 system, consists of a single guide RNA (sgRNA), which directs a Cas9 endonuclease to complementary target sites, and serves as a superior alternative to the previous protein-based systems. The techniques have been successfully applied to the development of better HIV-1 models, generation of protective mutations in endogenous/host cells, disruption of HIV-1 genomes and even reactivating latent viruses for better detection and clearance by host immune response. Here, we focus on gene editing-based HIV-1 treatment and research in addition to providing perspectives for refining these techniques.
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