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Cited by 77CRISPR and Genetic EngineeringVirus-based gene therapy researchHIV Research and Treatment

Newer Gene Editing Technologies toward HIV Gene Therapy

Open access
Manjunath, N., Yi, Guohua, Dang, Ying, Shankar, Premlata·2013·Viruses
DOI 10.3390/v5112748PMC PMC3856413
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Abstract

Despite the great success of highly active antiretroviral therapy (HAART) in ameliorating the course of HIV infection, alternative therapeutic approaches are being pursued because of practical problems associated with life-long therapy. The eradication of HIV in the so-called “Berlin patient” who received a bone marrow transplant from a CCR5-negative donor has rekindled interest in genome engineering strategies to achieve the same effect. Precise gene editing within the cells is now a realistic possibility with recent advances in understanding the DNA repair mechanisms, DNA interaction with transcription factors and bacterial defense mechanisms. Within the past few years, four novel technologies have emerged that can be engineered for recognition of specific DNA target sequences to enable site-specific gene editing: Homing Endonuclease, ZFN, TALEN, and CRISPR/Cas9 system. The most recent CRISPR/Cas9 system uses a short stretch of complementary RNA bound to Cas9 nuclease to recognize and cleave target DNA, as opposed to the previous technologies that use DNA binding motifs of either zinc finger proteins or transcription activator-like effector molecules fused to an endonuclease to mediate sequence-specific DNA cleavage. Unlike RNA interference, which requires the continued presence of effector moieties to maintain gene silencing, the newer technologies allow permanent disruption of the targeted gene after a single treatment. Here, we review the applications, limitations and future prospects of novel gene-editing strategies for use as HIV therapy.

AI snapshot

Written from the paper’s full text.

TL;DR

This review summarizes ZFN, TALEN, and CRISPR/Cas9 gene-editing technologies as potential HIV therapies, focusing on CCR5/CXCR4 disruption and proviral DNA excision. It argues these tools could permanently render cells HIV-resistant or eliminate latent provirus, but delivery, off-target effects, and cytotoxicity remain barriers.

Key findings

  • ZFN-mediated CCR5 disruption reached 50% in primary CD4+ T cells with <5% off-target effects at CCR2; CXCR4 disruption reached 30–34%, and adult HSCs achieved >25% CCR5 disruption.
  • TALEN and ZFN gave comparable CCR5 disruption (~45%) in 293T cells, but TALEN showed much lower cytotoxicity and significantly reduced off-target activity at the CCR2 locus.
  • CRISPR/Cas9 induced up to 18% CCR5 mutation in human cells in one study; the authors report unpublished data showing up to 50% CCR5 editing in 293T and Jurkat cells. A double-nicking strategy reduced off-target activity 50–1500 fold while maintaining on-target cleavage comparable to wild-type Cas9.
  • ZFN targeting of HIV proviral DNA could delete ~80% of proviral DNA in a computational model, and LTR-targeting ZFNs eradicated proviral DNA in cell lines and acutely/latently infected primary T cells.

Methods

  • Narrative review; no primary experimental design, sample, or statistical analysis is reported. It synthesizes published literature and clinical trial references on ZFN, TALEN, and CRISPR/Cas9 for HIV gene therapy.
  • Main analysis is a descriptive comparison of mechanisms, delivery strategies, specificity, and HIV applications, including CCR5/CXCR4 disruption and proviral DNA targeting.

Limitations

  • The paper states that delivery to relevant cell types remains a major bottleneck; TALEN’s larger size is a delivery hurdle; genome-wide off-target effects need careful evaluation; cytotoxicity in primary T cells and CD34+ cells needs study; latently infected cells are extremely rare (~one in 1 million CD4+ T cells), making in vivo delivery challenging; and clinical efficacy requires ART interruption, which is hard due to ethical and regulatory concerns.
  • Evident from the text: as a review, it does not provide new primary data except unpublished observations, and comparisons across studies are limited by different cell types, ZFN doses, and delivery methods—a point the paper itself notes for specificity evaluation.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.3390/v5112748

Cites

Statistics

Nothing to recompute.

Claims the paper makes

0 of 159 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    In unpublished studies, gRNAs can be selected to induce up to 50% gene editing at the CCR5 locus in 293T and Jurkat cells.

    “In unpublished studies, we have found that gRNAs can be selected to induce up to 50% gene editing at the CCR5 locus in 293 T, as well as in Jurkat cells.”
    Quote in the paperNot a substantial claim
  • Findingown data

    The authors successfully expressed both Cas9 and gRNA in a lentiviral vector for use in difficult-to-transfect primary T cells.

    “Moreover, we have also successfully expressed both Cas9 and gRNA in a lentiviral vector for use in difficult to transfect primary T cells.”
    Quote in the paperSubstantial claim
  • Premisecitation

    HAART does not offer a permanent cure because interruption of therapy leads to rapid rebound of viremia from latent reservoirs.

    “Moreover, although HARRT extends the lives of HIV-1 infected patients, it does not offer a permanent cure, since interruption of therapy leads to rapid rebound of viremia from latent reservoirs [3,4].”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [3] Finzi, 1999, Latent infection of CD4+ T cells provides a mechanism for lifelong persistence of HIV-1, even in patients on effective combination therapy, Nat. Med. source

      Not checked in the free run.

    • cites [4] Siliciano, 2003, Long-term follow-up studies confirm the stability of the latent reservoir for HIV-1 in resting CD4+ T cells, Nat. Med. source

      Not checked in the free run.

  • Premisenone given

    CCR5 is a major coreceptor for HIV.

    “Interest in this field began following the demonstration that CCR5 is a major coreceptor for HIV.”
    Quote in the paperSubstantial claim
  • Premisenone given

    Gene-editing methods allow permanent disruption of the selected gene(s).

    “On the other hand, gene-editing methods have attracted a lot of attention as potential therapy for HIV, as they allow permanent disruption of the selected gene(s).”
    Quote in the paperSubstantial claim
  • May combine two claims. 49
  • Classified as probably not a substantial claim. 10
  • The quote only partly states this claim. 1

  • May combine two claims.

    Premisecitation

    HAART is effective in suppressing viral replication and reducing viral loads in HIV patients.

    “Although highly active antiretroviral therapy (HAART) is effective in suppressing viral replication and reducing viral loads in HIV patients, it has limitations including high cost, patient compliance and side effects of long-term therapy, as well as emergence of drug resistance [1,2].”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [1] Carr, 2003, Toxicity of antiretroviral therapy and implications for drug development, Nat. Rev. Drug Discov. source

      Not checked in the free run.

    • cites [2] Zaccarelli, 2005, Multiple drug class-wide resistance associated with poorer survival after treatment failure in a cohort of HIV-infected patients, AIDS source

      Not checked in the free run.

  • May combine two claims.

    Premisenone given

    Following a double stranded break, DNA is repaired either by HR when a homologous DNA template is provided or by the error-prone NHEJ pathway, attended with small nucleotide additions or deletions that result in disruption of the reading frame and gene expression.

    “Now it has become clear that following double stranded break, DNA is repaired either by HR (when a homologous DNA template is also provided) or by the error prone non-homologous end joining (NHEJ) pathway, attended with small nucleotide additions or deletions that results in disruption of the reading frame and gene expression.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Most publications on HIV-1 gene therapy by ZFNs have focused on co-receptor CCR5 or CXCR4 disruption, but targeting the HIV-1 genome is a possible alternative strategy.

    “As for HIV-1 gene therapy by ZFNs, most publications have focused on co-receptor CCR5 or CXCR4 disruption [30,31,32], but targeting HIV-1 genome is a possible alternative strategy [33].”
    Quote in the paperNot a substantial claim0 of 4 sources checked
    • cites [30] Perez, 2008, Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases, Nat. Biotechnol. source

      Not checked in the free run.

    • cites [31] Wilen, 2011, Engineering HIV-resistant human CD4+ T cells with CXCR4-specific zinc-finger nucleases, PLoS Pathog. source

      Not checked in the free run.

    • cites [32] Yuan, 2012, Zinc-finger nuclease editing of human cxcr4 promotes HIV-1 CD4(+) T cell resistance and enrichment, Mol. Ther. source

      Not checked in the free run.

    • cites [33] Qu, 2013, Zinc-finger-nucleases mediate specific and efficient excision of HIV-1 proviral DNA from infected and latently infected human T cells, Nucleic Acids Res. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    The naturally occurring homozygous CCR5-Δ32 mutation results in a frame-shift mutation that disrupts CCR5 expression on the cell surface and confers resistance to HIV infection.

    “Naturally occurring homozygous CCR5-Δ32 mutation (32 bp deletion in the single coding exon of the gene), which results in a frame-shift mutation that disrupts CCR5 expression on the cell surface [7,8,9], confers resistance to HIV infection.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [7] Dean, 1996, Genetic restriction of HIV-1 infection and progression to AIDS by a deletion allele of the CKR5 structural gene, Science source

      Not checked in the free run.

    • cites [8] Liu, 1996, Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection, Cell source

      Not checked in the free run.

    • cites [9] Samson, 1996, Resistance to HIV-1 infection in caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene, Nature source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    RNA interference requires the continuous presence of siRNA and its gene ablation is never complete.

    “RNA interference has successfully been used to silence CCR5 as well as viral genes (reviewed in [12]); but it requires the continuous presence of siRNA, and the gene ablation is never complete.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [12] Subramanya, 2010, RNA interference-based therapeutics for human immunodeficiency virus HIV-1 treatment: Synthetic siRNA or vector-based shRNA?, Expert Opin. Biol. Ther. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    ZFNs have been widely utilized for genome editing in various types of cells and living organisms.

    “Taking advantage of the effective and sequence-specific gene modification capability of ZFNs, the technology has been widely utilized for genome editing in various types of cells and living organisms [23,24,25,26,27,28,29].”
    Quote in the paperNot a substantial claim0 of 7 sources checked
    • cites [23] Meng, 2008, Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases, Nat. Biotechnol. source

      Not checked in the free run.

    • cites [24] Carroll, 2008, Progress and prospects: Zinc-finger nucleases as gene therapy agents, Gene Ther. source

      Not checked in the free run.

    • cites [25] Doyon, 2008, Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases, Nat. Biotechnol. source

      Not checked in the free run.

    • cites [26] Shukla, 2009, Precise genome modification in the crop species Zea mays using zinc-finger nucleases, Nature source

      Not checked in the free run.

    • cites [27] Townsend, 2009, High-frequency modification of plant genes using engineered zinc-finger nucleases, Nature source

      Not checked in the free run.

    • cites [28] Jacob, 2010, Gene targeting in the rat: Advances and opportunities, Trends Genet. source

      Not checked in the free run.

    • cites [29] Urnov, 2010, Genome editing with engineered zinc finger nucleases, Nat. Rev. Genet. source

      Not checked in the free run.

  • May combine two claims.

    Premisereasoning

    A designed ZFN pair specifically recognizes 18-36 base pairs and can theoretically be designed to be unique and highly specific for the targeted gene.

    “Since each designed ZFN pair specifically recognizes 18–36 base pairs, it can theoretically be designed to make it unique and highly specific for the targeted gene.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    In most circumstances, NHEJ is the predominant repair mechanism for DSBs generated by ZFNs, and this repair pathway is attended with nucleotide insertions, deletions or frame-shift mutation leading to gene disruption.

    “However, the HR pathway needs an externally provided homologous DNA template; and thus in most circumstances, NHEJ is the predominant repair mechanism for DSBs generated by ZFNs. This repair pathway is attended with nucleotide insertions, deletions or frame-shift mutation [22], leading to gene disruption.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [22] Lieber, 2008, The mechanism of human nonhomologous DNA end joining, J. Biol. Chem. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Each zinc finger module of 30 amino acids recognizes three sequence-specific nucleotides, so a zinc finger module array recognizes between 9 and 18 base pairs.

    “The zinc finger domain typically contains three to six zinc finger modules linked together, and each finger module of 30 amino acids recognizes three sequence-specific nucleotides [18]. Therefore, each zinc finger module array recognizes between 9 and 18 base pairs.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [18] Pavletich, 1991, Zinc finger-DNA recognition: Crystal structure of a Zif268-DNA complex at 2.1 A, Science source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    HAART has limitations including high cost, patient compliance, side effects of long-term therapy, and emergence of drug resistance.

    “Although highly active antiretroviral therapy (HAART) is effective in suppressing viral replication and reducing viral loads in HIV patients, it has limitations including high cost, patient compliance and side effects of long-term therapy, as well as emergence of drug resistance [1,2].”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [1] Carr, 2003, Toxicity of antiretroviral therapy and implications for drug development, Nat. Rev. Drug Discov. source

      Not checked in the free run.

    • cites [2] Zaccarelli, 2005, Multiple drug class-wide resistance associated with poorer survival after treatment failure in a cohort of HIV-infected patients, AIDS source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    RNA interference has successfully been used to silence CCR5 as well as viral genes.

    “RNA interference has successfully been used to silence CCR5 as well as viral genes (reviewed in [12]); but it requires the continuous presence of siRNA, and the gene ablation is never complete.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [12] Subramanya, 2010, RNA interference-based therapeutics for human immunodeficiency virus HIV-1 treatment: Synthetic siRNA or vector-based shRNA?, Expert Opin. Biol. Ther. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Yuan and co-workers found that ZFN gene disruption was superior to shRNA in conferring protection against X4 tropic virus, and verified that CXCR4-ZFN disrupted CD4+ T cells conferred resistance in the humanized mouse model.

    “Similarly, Yuan and co-workers compared the efficiency of X4 targeting shRNAs and ZFN, and found that ZFN gene disruption was superior to shRNA in conferring protection against X4 tropic virus. They also verified that CXCR4-ZFN disrupted CD4+ T cells conferred resistance in the humanized mouse model [32].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [32] Yuan, 2012, Zinc-finger nuclease editing of human cxcr4 promotes HIV-1 CD4(+) T cell resistance and enrichment, Mol. Ther. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Infusion of ZFN-modified CD4+ T cells in vivo into NSG mice was not associated with any toxicity or T cell transformation.

    “Further, infusion in vivo into NSG mice was not associated with any toxicity or T cell transformation.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [41] Maier, 2013, Efficient clinical scale gene modification via zinc finger nuclease-targeted disruption of the HIV co-receptor CCR5, Hum. Gene Ther. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    When modified CXCR4-deficient CD4+ T cells were transplanted into NSG mice, the mice showed resistance to X4 HIV-1, but protection failed because of the emergence of CCR5-tropic virus mutants over time.

    “When the modified CD4+ T cells were transplanted into NSG mice, the mice showed resistance to X4 HIV-1. However, the protection failed because of the emergence of CCR5-tropic virus mutants over time [31].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [31] Wilen, 2011, Engineering HIV-resistant human CD4+ T cells with CXCR4-specific zinc-finger nucleases, PLoS Pathog. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Yao et al. showed that ZFN was able to efficiently and precisely disrupt CCR5 in embryonic and induced pluripotent stem cells, and that these cells could be differentiated into CD34+ HSCs.

    “Yao et al. showed that ZFN was able to efficiently and precisely disrupt CCR5 in embryonic and induced pluripotent stem cells (ESCs and iPSCs), and that these cells could be differentiated into CD34+ HSCs [42].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [42] Yao, 2012, Generation of CD34+ cells from CCR5-disrupted human embryonic and induced pluripotent stem cells, Hum. Gene Ther. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Transplantation of gene-modified HSCs in NSG mice resulted in multi-lineage reconstitution of human immune cell types, and the reconstituted cells showed resistance to HIV infection.

    “Transplantation of gene modified HSCs in NSG mice resulted in multi-lineage reconstitution of human immune cell types. Moreover the reconstituted cells showed resistance to HIV infection.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [43] Holt, 2010, Human hematopoietic stem/progenitor cells modified by zinc-finger nucleases targeted to CCR5 control HIV-1 in vivo, Nat. Biotechnol. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Individuals with mutations in the HIV co-receptor CCR5 have no major immune deficits except for a slight increase in the risk of West Nile infection.

    “On the other hand, individuals with mutations in the HIV co-receptor CCR5 have no major immune deficits [34,35] except for a slight increase in the risk of West Nile infection [36].”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [34] Melum, 2006, The 32-base pair deletion of the chemokine receptor 5 gene (CCR5-Delta32) is not associated with primary sclerosing cholangitis in 363 Scandinavian patients, Tissue Antigens source

      Not checked in the free run.

    • cites [35] Muntinghe, 2011, TNF-alpha levels are not increased in inflamed patients carrying the CCR5 deletion 32, Cytokine source

      Not checked in the free run.

    • cites [36] Telenti, 2009, Safety concerns about CCR5 as an antiviral target, Curr. Opin. HIV AIDS source

      Not checked in the free run.

  • May combine two claims.

    Interpretationcitation

    Following infection, gene-modified T cells were substantially enriched, probably because HIV killed the resident unmodified but not the modified T cells.

    “Moreover, following infection, the gene modified T cells were substantially enriched, probably because HIV killed the resident unmodified but not the modified T cells.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [30] Perez, 2008, Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases, Nat. Biotechnol. source

      Not checked in the free run.

  • May combine two claims.

    Interpretationcitation

    Disruption of CXCR4 or CCR5 alone may not be sufficient to provide stable protection as they could potentially force the emergence of the respective X4 or R5 tropic HIV-1 variants.

    “As these studies collectively show, disruption of CXCR4 or CCR5 alone may not be sufficient to provide stable protection as they could potentially force the emergence of the respective X4 or R5 tropic HIV-1 variants.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    Disruption of CCR5 and CXCR4 can only stop the spread of new virus, which could eventually result in a functional cure, but will not be sufficient to eradicate the virus from already infected cells.

    “As described above, disruption of CCR5 and CXCR4 can only stop the spread of new virus, which could eventually result in a functional cure. However, this will not be sufficient to eradicate the virus from already infected cells.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    After ex vivo expansion, ZFN-modified CD4+ T cells engrafted into NOG mice effectively reconstituted and provided significant resistance to subsequent HIV infection.

    “After ex vivo expansion, the modified CD4+ T cells were engrafted into NOG mice. Gene edited CD4+ T cells effectively reconstituted and provided significant resistance to subsequent HIV infection [30].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [30] Perez, 2008, Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases, Nat. Biotechnol. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    The modified adult HSCs could be reconstituted and were fully functional in NSG mice.

    “The modified adult HSCs could also be reconstituted and were fully functional in NSG mice [46].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [46] Li, 2013, Genomic editing of the HIV-1 coreceptor CCR5 in adult hematopoietic stem and progenitor cells using zinc finger nucleases, Mol. Ther. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    The CD4+ T-cell phenotype, cytokine production, and repertoire were comparable between ZFN-modified and control cells.

    “Moreover, the CD4+ T-cell phenotype, cytokine production, and repertoire were comparable between ZFN-modified and control cells.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [41] Maier, 2013, Efficient clinical scale gene modification via zinc finger nuclease-targeted disruption of the HIV co-receptor CCR5, Hum. Gene Ther. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Perez and colleagues demonstrated that ZFNs can permanently and specifically disrupt CCR5 in 50% of primary CD4+ T cells with minimal off-target effects (<5% at CCR2 loci).

    “Perez and colleagues demonstrated that ZFNs can permanently and specifically disrupt CCR5 in 50% of primary CD4+ T cells with minimal off target effects (<5% at CCR2 loci).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [30] Perez, 2008, Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases, Nat. Biotechnol. source

      Not checked in the free run.

  • May combine two claims.

    Premisenone given

    Early results suggest that infused gene-modified T cells are harmless, persist over time, and traffic to different organs.

    “Early results suggest that the infused gene modified T cells are harmless, persist over time, and traffic to different organs.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    CCR5 disruption by ZFN is heritable, and one-time treatment can potentially render the cell permanently HIV resistant.

    “Compared to conventional gene knockdown by RNAi, CCR5 disruption by ZFN is heritable and thus, one time treatment can potentially render the cell permanently HIV resistant.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Lambardo and colleagues disrupted CCR5 using a non-integrating lentiviral vector-delivered ZFN in HSCs, and AD5/35 delivered ZFN in primary T cells, human neural stem cells and iPSCs.

    “Likewise, Lambardo and colleagues also disrupted CCR5 using a non-integrating lentiviral vector-delivered ZFN in HSCs [44], and AD5/35 delivered ZFN in primary T cells, human neural stem cells (hNSCs) and in iPSCs [45].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [44] Lombardo, 2007, Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery, Nat. Biotechnol. source

      Not checked in the free run.

    • cites [45] Lombardo, 2011, Site-specific integration and tailoring of cassette design for sustainable gene transfer, Nat. Methods source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    The Doms group engineered CXCR4 deficient CD4+ T cells by CXCR4-ZFN delivery via AD5/35; the modified CD4+ T cells proliferated normally and showed resistance to CXCR4 tropic HIV-1 infection in vitro.

    “The Doms group at the University of Pennsylvania was the first to engineer CXCR4 deficient CD4+ T cells by CXCR4-ZFN delivery via AD5/35. The modified CD4+ T cells proliferated normally and showed resistance to CXCR4 tropic HIV-1 infection in vitro.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [31] Wilen, 2011, Engineering HIV-resistant human CD4+ T cells with CXCR4-specific zinc-finger nucleases, PLoS Pathog. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Wayengera reported that an 18 bp sequence from the HIV-pol gene could induce specific gene disruption, and found ZFN pairs that could delete ~80% of proviral DNA.

    “By using a computational model, Wayengera reported that an 18 bp sequence from the HIV-pol gene could induce specific gene disruption. They also used ZFN to target various sites within the proviral DNA and found the ZFN pairs that could delete ~80% of proviral DNA [52].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [52] Wayengera, 2011, Proviral HIV-genome-wide and pol-gene specific zinc finger nucleases: Usability for targeted HIV gene therapy, Theor. Biol. Med. Model source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Incubating CD4+ T cells with recombinant CCR5-ZFP protein led to gene disruption, although the efficiency was in the lower range of ~8%.

    “They recently incubated CD4+ T cells with recombinant CCR5-ZFP protein and found that this led to gene disruption, although the efficiency was in the lower range of ~8% [60].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [60] Gaj, 2012, Targeted gene knockout by direct delivery of zinc-finger nuclease proteins, Nat. Methods source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    TALEN technique has already been widely used for genome editing in a variety of cells, plants, model organisms, livestock and even human cells.

    “In a short time after discovery, this technique has already been widely used for genome editing in a variety of cells, plants, model organisms, livestock and even human cells [66,67].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [66] Joung, 2013, TALENs: A widely applicable technology for targeted genome editing, Nat. Rev. Mol. Cell Biol. source

      Not checked in the free run.

    • cites [67] Gaj, 2013, ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering, Trends Biotechnol. source

      Not checked in the free run.

  • May combine two claims.

    Interpretationcitation

    Concentration of ZFN is an important factor for off-target cleavage, suggesting that concentration of ZFN should be as low as possible.

    “Moreover, they also showed that concentration of ZFN is an important factor for off-target cleavage, suggesting that concentration of ZFN should be as low as possible [64].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [64] Pattanayak, 2011, Revealing off-target cleavage specificities of zinc-finger nucleases by in vitro selection, Nat. Methods source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisenone given

    TALE proteins contain a central domain with a variable number (5-30, average 15.5) of tandem monomer repeats that confer DNA binding specificity.

    “The central domain contains a variable number (5–30, average 15.5) of tandem monomer repeats that confer DNA binding specificity.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationcitation

    Taken together, non-viral delivery of ZFN is a feasible tool for HIV therapy, although further improvements may be required.

    “Taken together, these studies have shown that non-viral delivery of ZFN is a feasible tool for HIV therapy, although further improvements may be required.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [43] Holt, 2010, Human hematopoietic stem/progenitor cells modified by zinc-finger nucleases targeted to CCR5 control HIV-1 in vivo, Nat. Biotechnol. source

      Not checked in the free run.

    • cites [59] Cannon, P.M., Henley, J.E., Wood, T., Trong, L., Wang, J., Kim, K., Yan, J., Gregory, P.D., Lee, G., and Holmes, M.C. (2013, January 15–18). Electroporation of ZFN mRNA enables efficient CCR5 gene disruption in mobilized blood hematopoietic stem cells at clinical scale. Proceedings of the ASGCT 16th Annual Meeting, Salt Lake City, UT, USA. Abstract 183.

      Not checked in the free run.

    • cites [60] Gaj, 2012, Targeted gene knockout by direct delivery of zinc-finger nuclease proteins, Nat. Methods source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Analysis suggested that although off-target cleavage occurred only at consensus stretch of DNA, cleavage did not occur at all loci showing consensus predicted in silico.

    “This analysis suggested that although off-target cleavage occurred only at consensus stretch of DNA, cleavage did not occur at all loci showing consensus predicted in silico [65].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [65] Gabriel, 2011, An unbiased genome-wide analysis of zinc-finger nuclease specificity, Nat. Biotechnol. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S4 (based).

    Premisenone given

    Delivery of gene therapy vehicles to specific target cells has been a major bottleneck for translation to human therapy.

    “Delivery of gene therapy vehicles to specific target cells has been a major bottleneck for translation to human therapy.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Premisenone given

    Gene therapy for HIV infection is mainly focused on CD4+ T cells or CD34+ HSC.

    “Gene therapy for HIV infection is mainly focused on CD4+ T cells or CD34+ HSC.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationcitation

    Tight binding (complete homology) of one half can cause off-target effects even if the other half contains two-three mismatches, showing the importance of designing ZFNs with less binding energy at both arms to avoid off-target effects.

    “Thus, tight binding (complete homology) of one half can cause off target effects even if the other half contains two-three mismatches, showing the importance of designing ZFNs with less binding energy at both arms to avoid off-target effects.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [64] Pattanayak, 2011, Revealing off-target cleavage specificities of zinc-finger nucleases by in vitro selection, Nat. Methods source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    After engraftment into NSG mice, these cells supported multilineage reconstitution of human immune system, and the gene modified cells persisted even after 6 months of reconstitution.

    “After engraftment into NSG mice, these cells supported multilineage reconstitution of human immune system, and the gene modified cells persisted even after 6 months of reconstitution [59].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [59] Cannon, P.M., Henley, J.E., Wood, T., Trong, L., Wang, J., Kim, K., Yan, J., Gregory, P.D., Lee, G., and Holmes, M.C. (2013, January 15–18). Electroporation of ZFN mRNA enables efficient CCR5 gene disruption in mobilized blood hematopoietic stem cells at clinical scale. Proceedings of the ASGCT 16th Annual Meeting, Salt Lake City, UT, USA. Abstract 183.

      Not checked in the free run.

  • May combine two claims.

    Premisereasoning

    Because of the difficulty in targeting CD4+ T cells or CD34+ HSC in vivo, the general approach is to modify the cells in vitro with the idea that they may be used for reinfusion to infected subjects.

    “Because of the difficulty in targeting these cells in vivo, the general approach is to modify the cells in vitro with the idea that they may be used for reinfusion to infected subjects.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Lombardo et al. used a non-integrating lentivirus to deliver ZFN to disrupt CCR5 in CD34+ HSCs, with efficiency relatively low (5%).

    “With this in mind, Lombardo et al. used a non-integrating lentivirus to deliver ZFN to disrupt CCR5 in CD34+ HSCs. They were able to disrupt CCR5 in HSCs, though the efficiency was relatively low (5%) [44].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [44] Lombardo, 2007, Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery, Nat. Biotechnol. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Plasmid transfection by nucleofection resulted in 17% CCR5 disruption in HSC; when these cells were transplanted into NSG mice, the relatively low number of modified cells selectively expanded after in vivo HIV challenge.

    “Thus, plasmid transfection by nucleofection has been used for ZFN-mediated CCR5 disruption. This method resulted in 17% CCR5 disruption in HSC. When these cells were transplanted into NSG mice, the relatively low number of modified cells selectively expanded after in vivo HIV challenge [43].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [43] Holt, 2010, Human hematopoietic stem/progenitor cells modified by zinc-finger nucleases targeted to CCR5 control HIV-1 in vivo, Nat. Biotechnol. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    This CRISPR pathway is commonly used by bacteria to destroy foreign invaders like plasmids and phages, and to evade host innate immunity to enhance bacterial virulence.

    “This pathway is commonly used by bacteria to destroy foreign invaders (like plasmids and phages), and to evade host innate immunity to enhance bacterial virulence [84,85].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [84] Makarova, 2011, Evolution and classification of the CRISPR-Cas systems, Nat. Rev. Microbiol. source

      Not checked in the free run.

    • cites [85] Sampson, 2013, A CRISPR/Cas system mediates bacterial innate immune evasion and virulence, Nature source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Attempts to express TALEN in lentiviral vectors have failed, but it has been expressed successfully in adenoviral vectors.

    “However, although attempts to express TALEN in lentiviral vectors have failed, it has been expressed successfully in adenoviral vectors [82].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [82] Holkers, 2013, Differential integrity of TALE nuclease genes following adenoviral and lentiviral vector gene transfer into human cells, Nucleic Acids Res. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    A systematic comparison showed that the double nickase system had 50-1500 fold decreased off-target activity while maintaining on-target cleavage comparable to wild type Cas9.

    “Indeed, a systematic comparison of on and off-target cleavage showed that the double nickase system had 50–1500 fold decreased off-target activity while maintaining on target cleavage comparable to wild type Cas9.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [95] Ran, 2013, Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity, Cell source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Mussolino and co-workers compared CCR5 gene disruption using ZFN and TALEN side by side in 293 T cells, and comparable levels of gene disruption (~45%) could be achieved with both approaches.

    “Mussolino and co-workers compared CCR5 gene disruption using ZFN and TALEN side by side in 293 T cells. Comparable levels of gene disruption (~45%) could be achieved with both approaches.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [81] Mussolino, 2011, A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity, Nucleic Acids Res. source

      Not checked in the free run.

  • May combine two claims. The quote only partly states this claim.

    Premisecitation

    TALEN demonstrated much lower cytotoxicity and significantly reduced off-target activity at the CCR2 locus compared to ZFN.

    “However, TALEN demonstrated much lower cytotoxicity and significantly reduced off-target activity at the CCR2 locus.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [81] Mussolino, 2011, A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity, Nucleic Acids Res. source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Engineering crRNA and tracrRNA as a single gRNA enables easy gene editing with custom synthesized gRNA that can target any host gene.

    “This innovation enables easy gene editing with custom synthesized gRNA that can be used to target any host gene.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Wild type Cas9 cuts both strands of DNA, and it can be converted into single stranded DNA nickase by mutating one of the cleavage domains.

    “While wild type Cas9 cuts both strands of DNA, it can be converted into single stranded DNA nickase by mutating one of the cleavage domains.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [95] Ran, 2013, Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity, Cell source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Bacterial genomes contain CRISPR loci consisting of an array of short direct repeats of 21-47 nts (average 32) interspersed with short intervening spacers.

    “Bacterial genomes contain loci encoding what is known as clustered regularly interspaced palindromic repeats (CRISPR), consisting of an array of short direct repeats of 21–47 nts (average 32) interspersed with short intervening spacers (Figure 3).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [83] Wiedenheft, 2011, Structures of the RNA-guided surveillance complex from a bacterial immune system, Nature source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Transcribed CRISPR RNA (crRNA) products associate with Cas protein/nuclease and guide the complex to the target DNA that is complementary to the spacer sequence, whereupon the Cas protein cleaves the DNA to create DSB followed by DNA repair by NHEJ or HR.

    “The transcribed CRISPR RNA (crRNA) products associate with Cas protein/nuclease and guide the complex to the target DNA that is complementary to the spacer sequence, whereupon the Cas protein cleaves the DNA to create DSB followed by DNA repair by NHEJ or HR as described for ZFN and TALEN [83].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [83] Wiedenheft, 2011, Structures of the RNA-guided surveillance complex from a bacterial immune system, Nature source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    The Cas9 nickase mutant is safer to use with less off-target effects.

    “and the mutant is much safer to use with less off-target effects [88].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [88] Mali, 2013, RNA-guided human genome engineering via Cas9, Science source

      Not checked in the free run.

  • May combine two claims.

    Interpretationcitation

    Double nicking induces double stranded breaks in target DNA but is expected to induce only single stranded nicks in off-target genes because two adjacent off-target sites are rare, and these nicks are repaired by error-free BER.

    “While this induces double stranded breaks in target DNA, it is expected to induce only single stranded nicks in off-target genes (because of rarity of occurrence of two adjacent off-target sites) that get repaired by error free BER pathway.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [95] Ran, 2013, Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity, Cell source

      Not checked in the free run.

    • Rests on an assumption: Single stranded nicks are repaired by the error-free base excision repair (BER) pathway.
    • Rests on an assumption: Two adjacent off-target sites are rare.

  • May combine two claims.

    Interpretationreasoning

    A major advantage of CRISPR/Cas9 compared with other methods is that it requires much simpler design and a single cloning step, whereas other methods usually require labor-intensive design and screening.

    “Thus, a major advantage compared to other methods that usually require labor-intensive design and screening, is that the CRSPR/Cas9 requires much simpler design and a single cloning step.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The CRISPR locus is surrounded by a cohort of CRISPR-associated (Cas) genes.

    “The CRISPR locus is surrounded by a cohort of CRISPR-associated (Cas) genes.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [83] Wiedenheft, 2011, Structures of the RNA-guided surveillance complex from a bacterial immune system, Nature source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Interpretationreasoning

    Because CRISPR/Cas9 can target multiple regions to induce deletions, one potential application for HIV is to eliminate integrated proviral DNA as a strategy to eradicate reactivation from latently infected cells.

    “Since this system can also be used to target multiple regions to induce deletions, one potential application for HIV would be to eliminate the integrated proviral DNA as a strategy to eradicate reactivation from latently infected cells.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    These tools provide means to disrupt CCR5 and other host factors necessary for HIV infection as well as to delete the integrated HIV genome to eradicate HIV latency.

    “They provide powerful means to disrupt CCR5 and other host factors necessary for HIV infection as well as to delete the integrated HIV genome as a means to eradicate HIV latency.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: CCR5 and other host factors are necessary for HIV infection.

  • Classified as probably not a substantial claim.

    Interpretationreasoning

    ZFNs, TALENs and CRISPR/Cas9 are versatile tools for gene disruption that have the potential for revolutionizing the field of HIV gene therapy.

    “ZFNs, TALENs and CRISPR/Cas9 are versatile tools for gene disruption that have the potential for revolutionizing the field of HIV gene therapy.”
    Quote in the paperSubstantial claim
58 of 58

  • The paper relies on this without support.

    CCR5-Δ32 homozygotes are low frequency in the general population and suitable donors are difficult to identify.

    “However, due to the low frequency of CCR5-Δ32 homozygotes in the general population and the difficulties of identifying suitable donors, alternative methods to artificially disrupt CCR5 are being sought.”

  • The paper relies on this without support.

    The major viral receptor CD4 plays an important role in the immune system, which makes its deletion unacceptable.

    “However, the major viral receptor CD4 plays an important role in the immune system, which makes its deletion unacceptable.”

  • The paper relies on this without support.

    The CXCR4 receptor plays a physiologically important role, so its deletion in vivo may have functional consequences.

    “Further, the CXCR4 receptor plays a physiologically important role, raising concerns that its deletion in vivo may have functional consequences.”

  • The paper relies on this without support.

    Latently infected cells are extremely rare, approximately only one in 1 million CD4+ T cells.

    “For example, patients harbor the integrated HIV-1 provirus in resting T cells, and the latently infected cells are extremely rare (~only one in 1 million CD4+ T cells), so delivery of ZFN to these cells in vivo will pose a major challenge.”

  • The paper relies on this without support.

    The only prerequisite for DNA to be a CRISPR target site is that it needs to be preceded by an NGG sequence (PAM).

    “The only prerequisite for the DNA to be a CRISPR target site is that it needs to be preceded by a NGG sequence (protospacer adjacent motifs, PAM).”

  • The paper relies on this without support.

    Off-target cleavage occurs in a CRISPR/Cas9 dose-dependent manner.

    “Since the off-target cleavage occurs in a CRISPR/Cas9 dose-dependent manner, limiting the amount of Cas9 and gRNA plasmids delivered may be another way to minimize off-target effects.”

  • The paper relies on this without support.

    Single stranded nicks are repaired by the error-free base excision repair (BER) pathway.

    “Compared to DSB, which is repaired by error prone NHEJ pathway, single stranded nicks are repaired by error free base excision repair (BER) pathway.”

  • The paper relies on this without support.

    Two adjacent off-target sites are rare.

    “because of rarity of occurrence of two adjacent off-target sites”

  • The paper relies on this without support.

    CCR5 and other host factors are necessary for HIV infection.

    “They provide powerful means to disrupt CCR5 and other host factors necessary for HIV infection”
9 of 9

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Nuclease-based approaches have been used to disrupt HIV-1 entry coreceptors CCR5 or CXCR4 and proviral DNA-encoding viral proteins.

    “These approaches have been used to disrupt HIV-1 entry coreceptors C-C chemokine receptor 5 (CCR5) or C-C-C chemokine receptor 4 (CXCR4) and proviral DNA-encoding viral proteins”

    From RNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection (2014) · cites this paper as [9]

  • Engineered nucleases including ZFN, TALEN, and CRISPR Cas9 can disrupt target genes and have raised prospects of selectively deleting HIV-1 proviral DNA integrated into the host genome.

    “Advances in the engineered nucleases including zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR) associated 9 (Cas9) that can disrupt target genes have raised prospects of selectively deleting HIV-1 proviral DNA integrated into the host genome”

    From RNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection (2014) · cites this paper as [9]

The citation lists haven’t been fetched yet

References 96 · Cited by 77

Research works printed but not linked: 1

  1. Das, K.T., and Segal, D. (2012, January 22–27). Engineered Zinc Finger Nucleases to Inactivate the HIV Genome. Proceedings of the 19th HIV International Conference, Washington DC, USA. Abstract TUPEO21.

Authors
Manjunath, N., Yi, G., Dang, Y., & Shankar, P.
Affiliations
Texas Tech University Health Sciences Center (US)
Corresponding author
  • Manjunath, N.
  • Shankar, Premlata
ORCID
Venue
Viruses
Publisher
MDPI AG
Published
2013-11-14
Volume
5
Issue
11
Pages
2748-2766
ISSN
1999-4915 (ISSN-L)
DOI
10.3390/v5112748
PMC
PMC3856413
OpenAlex
W1989141229
Semantic Scholar
717c46c0970ef25f69ebeda5482cd65de17ed366
Cited by
77
References
96
Citations by year
2026: 2 · 2025: 1 · 2024: 3 · 2022: 4 · 2021: 3 · 2020: 3 · 2019: 7 · 2018: 2 · 2017: 7 · 2016: 14 · 2015: 19 · 2014: 12
FWCI
1.41
Type
journal article
Language
en
Open access
Licence
Locations
  • Viruses (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
  • National Heart, Lung, and Blood Institute (R21HL116268)
  • National Institute of Allergy and Infectious Diseases (R21AI108259, R01AI084795)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
  • Life Sciences › Immunology and Microbiology › Virology › HIV Research and Treatment
Keywords
HIV gene therapy, genome editing, CRISPR/Cas9, TALEN, zinc finger nucleases, homing endonucleases, CCR5, HIV eradication, bone marrow transplantation, DNA repair mechanisms, site-specific DNA cleavage, RNA interference
MeSH terms
Animals, DNA Repair, Humans, Genetic Therapy, HIV Infections, Clustered Regularly Interspaced Short Palindromic Repeats

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