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Genome editing-based HIV therapies Wan-Gang Gu Department of Immunology, Zunyi Medical University, Zunyi, Guizhou, 563000, China

Genome editing (GE)-based HIV therapy is achieved by modification of infection-related genes to produce HIVresistant cells followed by reinfusion of the modified cells into patients. The ultimate goal is to achieve a functional or actual cure for HIV infection. Despite multiple potential targets for GE-based HIV therapies, CCR5 is the most feasible owing to the naturally existing CCR5 d32 genotype which confers resistance to HIV. A recent clinical trial of infusion of modified autologous CD4+ T cells proved safety and efficacy within the limits of the studies. However, long-term evaluation of the safety and efficacy is required before GE-based HIV therapy is ready for clinical implementation. A therapy beyond highly-active antiretroviral therapy is needed Acquired immunodeficiency syndrome (AIDS; see Glossary) is a serious infectious disease characterized by the systemic collapse of the immune system caused by a retrovirus designated HIV-1. The pathogen was identified 2 years after the first report of the disease in 1981 [1]. The HIV-1 genome is composed of two identical copies of single-stranded RNA. The high mutation rate during the replication cycle results in a great likelihood for the development of drug resistance of the virus. More than 30 drugs have been developed for the clinical treatment of AIDS, and a substantial number of drug candidates are in clinical or preclinical trials, but there is no effective treatment to eliminate the virus from patients [2]. Although a preventive vaccine for HIV would be ideal, developing a successful preventive vaccine for HIV has been a long and complicated process. The only effective therapy used in clinical treatment for HIV infection is highly-active antiretroviral therapy (HAART), a combination of antiviral drugs targeting different steps in the virus life cycle [3]. However, an increasing number of patients have to face the problem that, due to the serious drug resistance, there are no suitable drugs to choose from for use in HAART for HIV infection. Scientists must develop alternative methods to combat the resistance of the virus to current therapies. HIV attacks mainly human CD4+ T lymphocytes, peripheral blood mononuclear cells (PBMC), monocytes, dendritic cells, and macrophages (Box 1). HIV infection is a complex interaction between virus and host. A substantial number of viral and host proteins are involved in this interaction. Corresponding author: Gu, W.-G. ([email protected], [email protected]). Keywords: HIV; AIDS; CCR5; genome editing; infusion. 0167-7799/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibtech.2014.12.006

Multiple proteins involved in the HIV life cycle have been validated for antiviral drug development. Theoretically, each step of HIV replication could be targeted for anti-HIV therapy (Box 1). The HIV-1 genome consists of nine genes, which are also important targets for anti-HIV research (Box 1). Developing molecular therapeutics for these targets could be viable, but GE-based therapies stand out as a possible alternative to HAART for the treatment of HIV infection. Numerous studies have been conducted to foster the development of successful GE-based therapies against HIV infection. In 2009, a patient was functionally cured of HIV infection by transplantation of allogeneic stem cells from a donor homozygous for the CCR5 d32 allele [4,5]. In the search for a 10/10 allele human leukocyte antigen (HLA)match donor for stem cell transplants, in most cases, more than one donor and occasionally more than 100 donors are available and the frequency of homozygosity for the CCR5 d32 allele is around 1% in Caucasians. There is thus a small, but reasonable chance of finding an HLA-matched donor homozygous for the CCR5 d32 allele [6]. Mimicking the natural homozygous CCR5 d32 by using GE technologies to induce the mutation is a viable choice. Glossary Acquired immunodeficiency syndrome (AIDS): a serious infectious disease characterized by the systemic collapse of the immune system caused by human immunodeficiency virus. Clustered regularly interspaced palindromic repeats (CRISPR): DNA loci contained in bacterial genomes. CRISPR consists of an array composed of 21–47 nucleotide (32 on average) short direct repeats with various intervening spacers. CRISPR is one component of the pathway used by bacteria to destroy foreign invaders. Highly-active antiretroviral therapy (HAART): the only effective treatment for controlling AIDS progression. It is a combination of drugs targeting different steps of virus infection. Hematopoietic stem and progenitor cells (HSPC): blood cells that can differentiate into all other blood cells, including myeloid and lymphoid lineages. Human induced pluripotent stem cells (hiPSCs): a type of pluripotent stem cells generated from adult cells by introducing specific genes. Human embryonic stem cells (hESCs): a type of pluripotent stem cells originating from the inner cell mass of blastocysts in the early preimplantation embryo. Transcription activator-like effectors (TALE): a class of DNA-binding proteins originating from Xanthomonas, a plant bacterial pathogen. The N and C termini of TALE proteins are responsible for localization and activation, while the central domain is responsible for binding to specific DNA sequences. Transcription activator-like effector nucleases (TALENs): a pair of DNA-binding domains from TALE is linked to the FokI endonuclease for binding to the target DNA at opposite sides. TALEs consist of multiple 33–35 amino acid repeats in which each repeat recognizes only one nucleotide. TALENs result in targeted double-stranded breaks which activate DNA damage response pathways. TALENs are widely used in various cell types and multiple organisms including humans. Zinc-finger nucleases (ZFNs): these nucleases comprise ZF motifs from ZF proteins linked to the nuclease domain of the restriction endonuclease Fok1. Each ZF module (30 amino acids) recognizes three sequence-specific nucleotides. Each ZF module array containing 3–6 ZF modules thus recognizes 9–18 bp. ZFNs are widely used in various cell types and multiple organisms including humans.

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Review The success of the stem cell transplant has encouraged the continued study of GE-based HIV therapies. Based on the findings of DNA repair studies [7], further studies on GEbased therapies against HIV infection have made significant progress in recent years. In this paper I outline why GE-based therapies are promising alternative treatments for HIV infection, and critically evaluate their limitations. The general concerns of GE-based HIV therapies include target selection, the choice of GE technology, and evaluation of safety and efficacy. Although there are many problems to be solved, GE-based therapies have the potential to be applied in clinical treatment in the future. Advances in GE-based therapies The technology that underpins GE is crucial for successful therapy, and will make future therapeutic advancements possible. Transcription activator-like effector nucleases (TALENs) [8–14], zinc-finger nucleases (ZFNs) [15–17], and the clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) systems have all been used in GE-based therapies (Table 1) [18–23]. The ZFN system has been the most widely used in GE studies targeting HIV infection [24]. Although there are only a few reports of GE-based HIV therapies using the TALEN and CRISPR/Cas 9 systems, these two GE tools may have great potential. The major strategy for GE-based HIV therapies is to produce engineered immune cells that are resistant to HIV infection or replication. The possible cell types for modification range from hematopoietic stem cells to dendritic cells and CD4+ T cells [25,26]. CD4+ T cells and CD34+ stem cells are the two groups of cells used most frequently for GE-based HIV therapies. The most frequently used method consists of two steps: modifying the cells in vitro then reinfusing the modified cells into patients (Figure 1) [27,28]. Multiple targets have been selected for GE-based therapies (Table 2). The current approaches are mainly classified into two categories: therapies targeting host genes involved in virus infection and replication, such as CCR5, and therapies introducing genes that interfere with HIV replication, such as host restriction factors and fusion inhibitors [28–30]. The first category plays a key role. Because CD4, the major receptor for HIV, is very important for the

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human immune system, deletion of the CD4 gene is lethal. Several clinical trials involving CD4 modification for HIV therapies have been conducted, but these studies are no longer being investigated for clinical treatment [31–33]. Although the CD4 receptor may not be an appropriate target, the co-receptors for viral entry are ideal targets for disruption [34]. CCR5 A naturally existing 32 bp deletion in the CCR5 gene leads to a non-functional receptor, which confers resistance to HIV strains that use CCR5. Cells that are naturally resistant to HIV are usually from donors with the CCR5 d32 genotype [35]. Consequently, multiple methods have been developed to construct engineered cells with non-functional CCR5. Since the first modification of CCR5 with ZFN, many studies have been carried out in this area [36,37]. ZFN-mediated modification of CCR5 in human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) demonstrated the potential for treatment of HIV infection [38]. Similar studies showed that mice treated with CCR5-disrupted hematopoietic stem or progenitor cells (HSPCs) achieved resistance to HIV infection [39,40]. Recently, an engineered CCR5 mutation near the d32 region was introduced by ZFNs into TZM-bl cells, which then exhibited resistance to multiple CCR5-tropic HIV strains [41]. Another study provides evidence for the safety of the permanent dysfunction of CCR5 by modification with ZFN (http://www.clinicaltrials.gov, Phase I by University of Pennsylvania, NCT00842634). In this study, 12 patients with chronic aviremic HIV infection received a single dose of autologous CD4 T cells modified by ZFN. Immune reconstitution and HIV resistance were evaluated by CD4 T cell count and viral RNA or DNA detection [42]. A significant increase in the CD4 T cell count was observed. Blood HIV DNA levels decreased in most patients. Viral RNA was undetectable in one patient (of four patients evaluated) [42]. Sangamo Biosciences has initiated a similar clinical trial (NCT01252641 and NCT01044654) [43]. Another clinical trial with modified CCR5 in autologous CD4+ T cells is ongoing (NCT01543152) [44]. Similarly, ZFN-modified CCR5 has been introduced into autologous hematopoietic stem cells for clinical trial [45]. CCR5 gene disruption by

Box 1. The HIV infection cycle and the HIV genome present multiple targets for anti-HIV therapies Co-receptor usage can shift in different stages of infection progression (Figure IA). T-tropic HIV uses CXCR4 as the co-receptor for infection of T lymphocytes at later stages of HIV infection, while Mtropic HIV uses CCR5 as the co-receptor for infection of macrophages, monocytes, peripheral blood mononuclear cells (PBMC), and T lymphocytes in early infection and throughout all stages of the infection [76,77]. M-tropic viruses are predominant in the early stages of infection. T-tropic viruses emerge when stable infection is established. Co-receptor usage may therefore shift from one to the other following therapies targeting one co-receptor. The HIV replication cycle (Figure IB) can be divided into the following stages: attachment and entry, reverse transcription, integration, protein translation, assembly, and release [78]. HIV infection begins with attachment to human cells. Initially, the viral gp120 binds to the CD4 receptor on the plasma membrane. The binding of gp120 to the CD4 molecule results in a conformational change in the viral 2

gp120 that exposes the co-receptor interaction domain. The subsequent interaction between gp120 and the co-receptor leads to fusion of the virus with the cytoplasm membrane [79], the final stage of virus entry into human cells. After entry, the HIV RNA is reverse transcribed into DNA to generate the provirus which is transported to the cell nucleus for integration. Provirus transport is a complex process that involves many host factors [80]. The integration of the provirus into the host genome is also a crucial step for HIV proliferation. The HIV genes located in the provirus are transcribed into mRNAs for protein translation, and the structural proteins that form the virus particle are translated in the cytoplasm [81]. The plasma membrane is the location of virus assembly, and newly assembled viruses are released through the plasma membrane [78]. The HIV-1 genome (Figure IC) consists of nine genes (Gag, Pol, Env, Vif, Vpr, Vpu, Tat, Rev, and Nef). A LTR (long terminal repeat) region is located at both extremities of the integrated HIV-1 genome [82].

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M-tropic HIV (R5)

T-tropic HIV (X4) CD4



PBMC Macrophages T lymphocytes Monocytes

T lymphocytes

Early stage

Stable stage

Infecon progression (B)

Infecon virus

Assembly and budding

New virus

CD4 Entry and uncoang


Viral RNA Reverse transcripon


Viral DNA Transcripon

Provirus Integraon Nucleus


Reading frame 1.






2. Pol

3. 0


5 kb




10 kb TRENDS in Biotechnology

Figure I. HIV infection, replication cycle and the HIV genome. (A) Co-receptor usage shift during different stages of infection progression. (B) The HIV replication cycle. (C) The HIV-1 genome.

ZFN and TALEN were compared by Mussolino and coworkers in 293 T cells [46]. The CRISPR/Cas9 system has been used for gene editing that targeted the human genome [47]. Recently, hiPSCs homozygous for the

CCR5 d32 mutation were produced using TALEN or CRISPR/Cas9 system in combination with piggyBac technology [48]. These engineered cells showed resistance to HIV-1 challenge when differentiated into monocytes/ 3

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Table 1. New GE technologiesa Nuclease ZFN

Origin Eukaryotes

Delivery tool Advantages AV, AAV, LV Compatible with various viral vectors; each ZF module (30 amino acids) recognizes three sequence-specific nucleotides; widely used in various cell types and multiple organisms including humans Xanthomonas AV Each TALE repeat (33–35 amino acids) recognizes TALEN one nucleotide; widely used in various cell types and multiple organisms including humans Plasmids RNA-guided DNA endonuclease; targets multiple CRISPR/Cas Bacteria sites simultaneously to produce large gene fragment deletions; much simpler design (a short RNA for the gene of interest); potential application in various hosts including humans

Disadvantages Design of ZF arrays for sequence recognition is required; off-target effects

Design of TALE arrays for sequence recognition is required; the binding site starts with a T base; larger size than ZFN; off-target effects The target sequence needs to be preceded by PAM; off-target effects


AV, Adenovirus; AAV, adeno-associated virus; CRISPR/Cas, clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9; LV, lentivirus; PAM, protospacer adjacent motifs; TALE, transcription activator-like effector; TALEN, transcription activator-like effector nuclease; ZFN, zinc-finger nuclease.

macrophages. This may provide an approach to functionally cure of AIDS. Other studies showed that GE of the CCR5 locus, by incorporating multiple restriction factors (APOBEC3G, TRIM5a, D128K, or Rev M10), leads to resistance to both CCR5- and CXCR4-using viruses [28]. CXCR4 Although debatable, disruption of the C-X-C chemokine receptor type 4 gene (CXCR4) with ZFN did confer resistance to T-tropic HIV in several studies [49,50]. Upon disruption of CXCR4 with ZFNs, human T cells exhibited resistance to X4-tropic HIV-1 strains. The loss of CXCR4 also conferred resistance to X4 HIV-1 in humanized mouse, although the resistance was lost when R5-tropic viruses emerged [49]. In another study, disruption of CXCR4 with ZFNs in CD4+ T cells provided protection from HIV-1 infection in tissue culture and in mice. Moreover, after engraftment of engineered T cells, HIV-1-infected mice had lower viral load [50]. Considering the important roles of

CCR5 (Edited)

CCR5 (Wild type) Genome eding (ZFN,TALEN,CRISPR/Cas9 system)

CD4+ T cells CD34+ stem cells

CD4+ T cells CD34+ stem cells



Paent TRENDS in Biotechnology

Figure 1. Modification of CCR5 for treatment of HIV infection. CD4+ T cells or CD34+ stem cells are isolated from HIV infected patient by apheresis. Then the cells are modified with genome editing tools (ZFN, TALEN or CRISPS/Cas9 system) to produce HIV-resistant cells with CCR5 mutation. The HIV-resistant cells are then reinfused into the patient for treatment of HIV infection [39,42].


CXCR4 in normal physiology, autologous transplantation with CXCR4-disrupted cells will need to be deferred until long-term safety has been fully evaluated in animals. Or is it possible to find a safe mode of disruption of CXCR4? Proviral DNA Eliminating the HIV proviral DNA from infected cells with GE technologies is another novel strategy. The incurability of HIV infection can be attributed to the integration of viral DNA into the human genome and the persistence of proviral DNA. How to eliminate the proviral DNA from infected cells has been a confusing issue to scientists. Up to 80% of integrated viral DNA can be removed from the human genome with ZFNs targeting the proviral HIV Pol gene or the whole proviral genome [51]. Three ZFNs were also designed to cleave the conserved regions of Gag, Pol, and Rev [52]. The full-length proviral DNA can also be excised from human genome with ZFNs targeting conserved regions within HIV-1 50 and 30 long terminal repeats (LTRs). The excision frequency reached 45.9% in human cell lines infected with HIV-1 [53]. The LTRs target sites were efficiently cleaved and mutated by the CRISPR/Cas9 system. The CRISPR/Cas9 system was also able to delete internal HIV genes from a human chromosome [54]. Highlyspecific targets were identified and efficiently edited by Cas9 system with guide RNAs (gRNA) homologous to sequences within the LTR U3 region of HIV-1, without activation of virus gene expression or viral replication in latently infected cells (microglial, promonocytic, and T cells). A 9709 bp fragment of proviral DNA spanning the 50 to 30 LTRs was excised completely from the human genome. No detectable genotoxicity or off-target editing were found. Moreover, HIV-1 infection was prevented in the presence of multiplex gRNAs in cells expressing Cas9 [55]. ZFNs have also been designed to target proviral DNA of other viruses including HTLV-1 [56], HSV-2 [57], and HBV [58]. Although the above trials are exciting, and proviral DNA remains an appealing target for GE-based therapies, one major challenge in clinical treatment is how to deliver the ZFNs or CRISPR/Cas9 system to the latently infected cells accurately because these cells are extremely rare (one in 1 million CD4+ cells) [18].

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Table 2. Summary of recent GE-based HIV therapiesa Target gene CCR5 CCR5 CCR5 CCR5 CCR5 CCR5 CCR5 CCR5 CCR5 CCR5 CCR5 CCR5 (RF) CXCR4 CXCR4 Provirus (LTR) Provirus (Pol) Provirus (LTR) Provirus (LTR) LEDGF/p75 TSPO


Target Cell Autologous CD4+ T cells (clinical trial) Autologous CD4+ T cells (clinical trial) Autologous CD4+ T cells (clinical trial) Autologous hematopoietic stem cells (clinical trial) Induced pluripotent stem cells, embryonic stem cells Hematopoietic stem, progenitor cells Hematopoietic stem, progenitor cells TZM-bl cells 293 T cells 293 T cells iPSCs Jurkat cells, JLTRG-R5 cells T cells T cells Infected T cells TZM-bl cells, HeLa-derived JC53-BL cells Infected T cells Microglial, promonocytic, and T cells 293 T cells, Jurkat cells 293 T cells

Refs [42] [43] [44] [45] [38] [39] [40] [41] [46] [47] [48] [28] [49] [50] [53] [51] [54] [55] [61] [62]


Abbreviations: CCR5, C-C chemokine receptor type 5; CCR5 (RF), restriction factors inserted into the CCR5 locus; CXCR4, C-X-C chemokine receptor type 4; CD4, cluster of differentiation 4; CRISPR/Cas, clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9; LEDGF, lens epithelium-derived growth factor; LTR, long terminal repeat; TALEN, transcription activator-like effector nuclease; TSPO, mitochondrial translocator protein; ZFN, zinc-finger nuclease.

Other targets Lens epithelium-derived growth factor (LEDGF)/p75, also known as PC4 and SFRS1 interacting protein 1 (PSIP1), is the first well-characterized cellular cofactor for HIV-1 integrase [59]. It plays a crucial role in viral integration, tethering the pre-integration complex (PIC) to the nucleus through interaction with integrase [60]. Similarly to the case of CCR5, in CD4+ T cells, LEDGF depletion is well tolerated. In recent studies, all LEDGF expression was completely eradicated with TALENs. The deletion of whole LEDGF gene or the exons encoding the integrase binding domain (IBD) resulted in inhibition of viral integration and impaired virus replication in human cell lines [61]. Now that LEDGF/p75 has been validated as a potential therapeutic target, this study may provide an approach for therapeutic treatment of HIV infection. Another potential target might be the mitochondrial translocator protein (TSPO), which was found to inhibit HIV-1 Env glycoprotein expression [62]. Although the CRISPR/Cas9 system was only used to generate a TSPO knockout in 293T cells for this study, the locus could be an attractive target for other GE-based strategies such as CRISPR/Cas9-mediated transcriptional activation [23]. Many other targets in the HIV life cycle can also be chosen for GE-based HIV therapies. The hope is that HIV infection could be cured or controlled with a single infusion of HIV-resistant cells in the future. Concerns in current GE-based therapies Although great progress has been achieved in GE basedtherapies, and many have proceeded into clinical trials [22–25], several issues give cause for concern. Safety is a fundamental concern in GE-based therapies. Although the recent advance in ZFN-modified analogous CD4+ T cell infusion has provided some proof of the safety of this therapy, a long-term safety evaluation is needed. It has

been proposed to conduct trials of this therapy with a larger population of HIV-infected patients. In addition, one serious adverse event attributed to a transfusion reaction was observed in an ongoing clinical trial [42], and this should be investigated. It is necessary to determine whether this is an occasional event or a regular event in this type of transfusion. If it is a regular event, then how frequent is it? How might it be avoided or prevented? A series of indices, including viral RNA, viral DNA, and CD4 counts, have been selected to evaluate the efficacy of the therapy. Although the data are promising, it is too early to determine if the goal of the therapy will be to cure the HIV infection or control the progression of AIDS. Although the ‘Berlin patient’ was functionally cured of HIV infection after stem cell transplantation, the molecular mechanism of a functional cure is not yet clear. A mechanistic approach may provide valuable insight into understanding viral infection and could facilitate the next generation of GE-based therapies. The success of the ‘Berlin patient’ was also dependent on finding the appropriate stem cell donor for transplantation [4,5]. The difficulties in finding HLA-matched donors with homozygosity for the CCR5 d32 deletion, as well as the problems in the transplant process, undercut the feasibility of deploying this therapy. Because of the success in using HIV-resistant GE stem cells in some studies [34,39,63], transplanting GE autologous stem cells may be a feasible method for clinical treatment once sufficient evidence has been gathered from clinical trials [45,64,65]. Another concern could be the choice of target for these therapies. CCR5 serves as the predominant co-receptor in the early stage of HIV infection, and CXCR4 plays a key role as co-receptor when stable infection is established. HIV co-receptor usage can switch between CCR5 and CXCR4 (Box 1), resulting in failure of protection if therapies target only one of these co-receptors [49]. A small 5

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proportion of CXCR4-using HIV was detected via ultradeep sequencing before stem cell transplantation in the ‘Berlin patient’ [4]. For unknown reasons, these X4 variants did not rebound after transplantation, which has confounded researchers. Co-receptor usage shift is an important problem in anti-HIV research, and should be kept in mind when designing GE-based therapies in the future. A technical concern for GE tools is off-target cleavage events, which can lead to unintended disruption of physiologically important genes, and can have unpredictable consequences. High-throughput methods have been applied to comprehensively profile the off-target cleavage sites [41,66,67], and predicting possible off-target cleavage sites and understanding their effects will greatly improve the efficiency and the safety of GE-based therapies.

human stem cells [68]. High-throughput technologies may contribute to wider evaluation of off-target events in GE-based therapies [75]. As we have discussed, in addition to CCR5, other targets including CXCR4, proviral DNA, and LEDGF have been selected for GE-based therapies. With the development of GE technologies and better understanding of the HIV infection process, more targets may be approached. Although there are several important issues to be addressed, significant advances have been made and the results from the recent clinical trial are especially exciting. Although the viral load can be reduced to undetectable levels with HAART under the appropriate supervision, it is impossible to erase the virus from patients undergoing traditional therapy. However, because GE-based HIV therapies employ fundamentally different strategies, patients may be functionally cured in the future.

Concluding remarks and future perspectives The current GE-based HIV therapies are mainly based on reinfusion of modified CD4+ T lymphocytes or CD34+ stem cells. CCR5 is the major current focus for two reasons: naturally existing CCR5 d32 confers resistance to HIV infection, and one patient was cured of HIV infection by transplanting stem cells from a donor homozygous for the CCR5 d32 allele. Production of HIV-resistant cells with an artificial CCR5 mutation and reinfusing the cells into patients to confer HIV resistance is the most common strategy. Both safety and long term efficacy are major concerns for GE-based therapies. In the current studies, most attention was paid to the pre-infusion procedures including selection of GE technologies [18,19], delivery tools, and cell types. Although results from clinical trial are promising, according to a series of indices including viral RNA, viral DNA and CD4 count, post-infusion evaluation is a long-term and systematic project. Only the post-infusion evaluation results can tell us how far the GE-based HIV therapies will reach. Moreover, several important scientific problems remain to be addressed (Box 2). Is modification of CCR5 sufficient for controlling viral infection when viruses targeting CXCR4 are present in the population? If the answer is yes, then what is the mechanism? Another important issue concerns off-target events [68–72]. The off-target events should be fully evaluated before clinical application. Recently a novel tool, TALENoffer, has been used for genomewide prediction of TALEN off-target sites [73]. An offtarget search tool for Cas9/gRNA, CasOT, has also been developed [74]. CRISPR-Cas9- and TALEN-induced offtarget mutations were also systematically assessed in


Box 2. Outstanding questions  Is modification of CCR5 sufficient for controlling of the virus infection when CXCR4 using virus exists? If the answer is yes, then what is the mechanism?  Is there a safe mode for CXCR4 modification to control HIV infection without disruption of normal physiological function?  Is it possible to target CCR5 and CXCR4 together for genome editing based HIV therapies? 6

The author was supported by the National Natural Science Foundation of China (81360503), the United Foundation of Guizhou (Qiankehe J LKZ [2013] 21), the Incubation Project for 2011 Collaborative Innovation Center for Tuberculosis Prevention and Cure in Guizhou Province, the West Light Talent Training Plan of the Chinese Academy of Sciences, and the grant from Zunyi Medical University for newly recruited talents to W-G.G.

References 1 Centers for Disease Control and Prevention (2011) HIV surveillance – United States, 1981–2008. MMWR Morb. Mortal. Wkly. Rep. 60, 689–693 2 Gu, W.G. et al. (2014) Anti-HIV drug development through computational methods. AAPS J. 16, 674–680 3 Gilden, D. (1996) When HAART is not enough. GMHC Treat. Issues 10, 1–6 4 Hutter, G. et al. (2009) Long-term control of HIV by CCR5 Delta32/ Delta32 stem-cell transplantation. N. Engl. J. Med. 360, 692–698 5 Allers, K. et al. (2010) Evidence for the cure of HIV infection by CCR5Delta32/Delta32 stem cell transplantation. Blood 117, 2791– 2799 6 Burke, B.P. et al. (2014) CCR5 as a natural and modulated target for inhibition of HIV. Viruses 6, 54–68 7 Wyman, C. and Kanaar, R. (2006) DNA double-strand break repair: all’s well that ends well. Annu. Rev. Genet. 40, 363–383 8 Osborn, M.J. et al. (2013) TALEN-based gene correction for epidermolysis bullosa. Mol. Ther. 21, 1151–1159 9 Li, T. and Yang, B. (2013) TAL effector nuclease (TALEN) engineering. Methods Mol. Biol. 978, 63–72 10 Bedell, V.M. et al. (2012) In vivo genome editing using a high-efficiency TALEN system. Nature 491, 114–118 11 Berdien, B. et al. (2014) TALEN-mediated editing of endogenous T-cell receptors facilitates efficient reprogramming of T lymphocytes by lentiviral gene transfer. Gene Ther. 21, 539–548 12 Ding, Q. et al. (2014) A TALEN genome-editing system for generating human stem cell-based disease models. Cell Stem Cell 12, 238–251 13 Katsuyama, T. et al. (2013) An efficient strategy for TALEN-mediated genome engineering in Drosophila. Nucleic Acids Res. 41, e163 14 Heigwer, F. et al. (2013) E-TALEN: a web tool to design TALENs for genome engineering. Nucleic Acids Res. 41, e190 15 Anguela, X.M. et al. (2013) Robust ZFN-mediated genome editing in adult hemophilic mice. Blood 122, 3283–3287 16 Gupta, A. et al. (2012) An optimized two-finger archive for ZFNmediated gene targeting. Nat. Methods 9, 588–590 17 Mattis, A.N. and Willenbring, H. (2012) A ZFN/piggyBac step closer to autologous liver cell therapy. Hepatology 55, 2033–2035 18 Manjunath, N. et al. (2013) Newer gene editing technologies toward HIV gene therapy. Viruses 5, 2748–2766 19 Gaj, T. et al. (2013) ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol. 31, 397–405

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Review 20 Holkers, M. et al. (2013) Differential integrity of TALE nuclease genes following adenoviral and lentiviral vector gene transfer into human cells. Nucleic Acids Res. 41, e63 21 Montague, T.G. et al. (2014) CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing. Nucleic Acids Res. 42, W401– W407 22 Shao, Y. et al. (2014) CRISPR/Cas-mediated genome editing in the rat via direct injection of one-cell embryos. Nat. Protoc. 9, 2493–2512 23 Gilbert, L.A. et al. (2014) Genome-scale CRISPR-mediated control of gene repression and activation. Cell 159, 647–661 24 Owens, B. (2014) Zinc-finger nucleases make the cut in HIV. Nat. Rev. Drug Discov. 13, 321–322 25 Rossetti, M. et al. (2012) HIV-derived vectors for gene therapy targeting dendritic cells. Adv. Exp. Med. Biol. 762, 239–261 26 Merkert, S. et al. (2010) Efficient ZFN-based gene inactivation in transgenic human iPS cells as a model for gene editing in patientspecific cells. J. Stem Cells Regen. Med. 6, 118 27 van Lunzen, J. et al. (2007) Transfer of autologous gene-modified T cells in HIV-infected patients with advanced immunodeficiency and drug-resistant virus. Mol. Ther. 15, 1024–1033 28 Voit, R.A. et al. (2013) Generation of an HIV resistant T-cell line by targeted ‘stacking’ of restriction factors. Mol. Ther. 21, 786–795 29 Kiem, H.P. et al. (2012) Hematopoietic-stem-cell-based gene therapy for HIV disease. Cell Stem Cell 10, 137–147 30 Chan, E. et al. (2014) Gene therapy strategies to exploit TRIM derived restriction factors against HIV-1. Viruses 6, 243–263 31 Mitsuyasu, R.T. et al. (2000) Prolonged survival and tissue trafficking following adoptive transfer of CD4zeta gene-modified autologous CD4+ and CD8+ T cells in human immunodeficiency virus-infected subjects. Blood 96, 785–793 32 Walker, R.E. et al. (2000) Long-term in vivo survival of receptormodified syngeneic T cells in patients with human immunodeficiency virus infection. Blood 96, 467–474 33 Deeks, S.G. et al. (2002) A phase II randomized study of HIV-specific T-cell gene therapy in subjects with undetectable plasma viremia on combination antiretroviral therapy. Mol. Ther. 5, 788–797 34 Zhang, J. et al. (2012) Why CCR5 is chosen as the target for stem cell gene therapy for HIV infection? J. Nanosci. Nanotechnol. 12, 2045– 2048 35 Nkenfou, C.N. et al. (2013) Distribution of CCR5-Delta32, CCR5 promoter 59029 A/G, CCR2-64I and SDF1-30 A genetic polymorphisms in HIV-1 infected and uninfected patients in the west region of Cameroon. BMC Res. Notes 6, 288 36 Perez, E.E. et al. (2008) Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. Nat. Biotechnol. 26, 808–816 37 Wang, J. et al. (2014) Targeted gene addition to a predetermined site in the human genome using a ZFN-based nicking enzyme. Genome Res. 22, 1316–1326 38 Yao, Y. et al. (2011) Generation of CD34+ cells from CCR5-disrupted human embryonic and induced pluripotent stem cells. Hum. Gene Ther. 23, 238–242 39 Holt, N. et al. (2010) Human hematopoietic stem/progenitor cells modified by zinc-finger nucleases targeted to CCR5 control HIV-1 in vivo. Nat. Biotechnol. 28, 839–847 40 Li, L. et al. (2013) Genomic editing of the HIV-1 coreceptor CCR5 in adult hematopoietic stem and progenitor cells using zinc finger nucleases. Mol. Ther. 21, 1259–1269 41 Badia, R. et al. (2014) Gene editing using a zinc-finger nuclease mimicking the CCR5Delta32 mutation induces resistance to CCR5using HIV-1. J. Antimicrob. Chemother. 69, 1755–1759 42 Tebas, P. et al. (2014) Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV. N. Engl. J. Med. 370, 901–910 43 ClinicalTrials.gov (2014) Phase 1 dose escalation study of autologous T-cells genetically modified at the CCR5 gene by zinc finger nucleases in HIV-infected patients. Published online June 4, 2014. http:// clinicaltrials.gov/ct2/show/NCT01044654/ 44 ClinicalTrials.gov (2014) Dose escalation study of cyclophosphamide in HIV-infected subjects on HAART receiving SB-728-T. Published online August 22, 2014. http://clinicaltrials.gov/ct2/show/NCT01543152 45 California Stem Cell Agency (2013) Zinc finger nuclease-based stem cell therapy for AIDS. http://www.cirm.ca.gov/our-progress/awards/ ziinc-finger-nuclease-based-stem-cell-therapy-aids

Trends in Biotechnology xxx xxxx, Vol. xxx, No. x

46 Mussolino, C. et al. (2011) A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity. Nucleic Acids Res. 39, 9283–9293 47 Cho, S.W. et al. (2013) Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat. Biotechnol. 31, 230–232 48 Ye, L. et al. (2014) Seamless modification of wild-type induced pluripotent stem cells to the natural CCR5Delta32 mutation confers resistance to HIV infection. Proc. Natl. Acad. Sci. U.S.A. 111, 9591–9596 49 Wilen, C.B. et al. (2011) Engineering HIV-resistant human CD4+ T cells with CXCR4-specific zinc-finger nucleases. PLoS Pathog. 7, e1002020 50 Yuan, J. et al. (2012) Zinc-finger nuclease editing of human cxcr4 promotes HIV-1 CD4+ T cell resistance and enrichment. Mol. Ther. 20, 849–859 51 Wayengera, M. (2011) Proviral HIV-genome-wide and pol-gene specific zinc finger nucleases: usability for targeted HIV gene therapy. Theor. Biol. Med. Model. 8, 26 52 Das, K.T.S. and Segal, D. (2014) Engineered zinc finger nucleases to inactivate the HIV genome. In Proceedings of the 19th HIV International Conference. Washington DC, USA, 22–27 July 2012, Abstract TUPEO21 (http://pag.aids2012.org/Abstracts.aspx?AID=5906) 53 Qu, X. et al. (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. 41, 7771–7782 54 Ebina, H. et al. (2013) Harnessing the CRISPR/Cas9 system to disrupt latent HIV-1 provirus. Sci. Rep. 3, 2510 55 Hu, W. et al. (2014) RNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection. Proc. Natl. Acad. Sci. U.S.A. 111, 11461–11466 56 Tanaka, A. et al. (2013) A novel therapeutic molecule against HTLV-1 infection targeting provirus. Leukemia 27, 1621–1627 57 Wayengera, M. (2011) Identity of zinc finger nucleases with specificity to herpes simplex virus type II genomic DNA: novel HSV-2 vaccine/ therapy precursors. Theor. Biol. Med. Model. 8, 23 58 Cradick, T.J. et al. (2010) Zinc-finger nucleases as a novel therapeutic strategy for targeting hepatitis B virus DNAs. Mol. Ther. 18, 947–954 59 Demeulemeester, J. et al. (2014) LEDGINs, non-catalytic site inhibitors of HIV-1 integrase: a patent review (2006–2014). Expert Opin. Ther. Pat. 24, 609–632 60 Christ, F. and Debyser, Z. (2012) The LEDGF/p75 integrase interaction, a novel target for anti-HIV therapy. Virology 435, 102–109 61 Fadel, H.J. et al. (2014) TALEN knockout of the PSIP1 gene in human cells: analyses of HIV-1 replication and allosteric integrase inhibitor mechanism. J. Virol. 88, 9704–9717 62 Zhou, T. et al. (2014) The mitochondrial translocator protein, TSPO, inhibits HIV-1 envelope glycoprotein biosynthesis via the endoplasmic reticulum-associated protein degradation pathway. J. Virol. 88, 3474– 3484 63 Didigu, C. and Doms, R. (2014) Gene therapy targeting HIV entry. Viruses 6, 1395–1409 64 Trounson, A. et al. (2011) Clinical trials for stem cell therapies. BMC Med. 9, 52 65 Lutz, S.E. et al. (2014) Stem cell-based therapies for multiple sclerosis: recent advances in animal models and human clinical trials. Regen. Med. 9, 129–132 66 Gabriel, R. et al. (2011) An unbiased genome-wide analysis of zincfinger nuclease specificity. Nat. Biotechnol. 29, 816–823 67 Pattanayak, V. et al. (2011) Revealing off-target cleavage specificities of zinc-finger nucleases by in vitro selection. Nat. Methods 8, 765–770 68 Veres, A. et al. (2014) Low incidence of off-target mutations in individual CRISPR-Cas9 and TALEN targeted human stem cell clones detected by whole-genome sequencing. Cell Stem Cell 15, 27–30 69 Hruscha, A. et al. (2013) Efficient CRISPR/Cas9 genome editing with low off-target effects in zebrafish. Development 140, 4982–4987 70 Cradick, T.J. et al. (2011) ZFN-site searches genomes for zinc finger nuclease target sites and off-target sites. BMC Bioinformatics 12, 152 71 Lin, Y. et al. (2014) CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences. Nucleic Acids Res. 42, 7473–7485 72 Shen, B. et al. (2014) Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Nat. Methods 11, 399–402 73 Grau, J. et al. (2013) TALENoffer: genome-wide TALEN off-target prediction. Bioinformatics 29, 2931–2932 7

TIBTEC-1229; No. of Pages 8

Review 74 Xiao, A. et al. (2014) CasOT: a genome-wide Cas9/gRNA off-target searching tool. Bioinformatics Published online January 21, 2014. (http://dx.doi.org/10.1093/bioinformatics/btt764) 75 Pattanayak, V. et al. (2013) High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat. Biotechnol. 31, 839–843 76 Loftin, L.M. et al. (2011) R5X4 HIV-1 coreceptor use in primary target cells: implications for coreceptor entry blocking strategies. J. Transl. Med. 9 (Suppl. 1), S3 77 Naif, H.M. (2014) Pathogenesis of HIV Infection. Infect. Dis. Rep. 5, e6 78 Sundquist, W.I. and Krausslich, H.G. (2012) HIV-1 assembly, budding, and maturation. Cold Spring Harb. Perspect. Med. 2, a006924


Trends in Biotechnology xxx xxxx, Vol. xxx, No. x

79 Kassler, K. and Sticht, H. (2013) Molecular mechanism of HIV-1 gp120 mutations that reduce CD4 binding affinity. J. Biomol. Struct. Dyn. 32, 52–64 80 Di Primio, C. et al. (2013) Single-cell imaging of HIV-1 provirus (SCIP). Proc. Natl. Acad. Sci. U.S.A. 110, 5636–5641 81 Maiuri, P. et al. (2010) Real-time imaging of the HIV-1 transcription cycle in single living cells. Methods 53, 62–67 82 Hadi, K. et al. (2013) A mutagenesis approach for the study of the structure–function relationship of human immunodeficiency virus type 1 (HIV-1) Vpr. In Genetic Manipulation of DNA and Protein – Examples from Current Research (Figurski, D., ed.), InTech Chapter 10 In: http:// dx.doi.org/10.5772/45824)

Genome editing-based HIV therapies.

Genome editing (GE)-based HIV therapy is achieved by modification of infection-related genes to produce HIV-resistant cells followed by reinfusion of ...
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