Application of gene-editing technologies to HIV-1

Drake, Mary Jane; Bates, Paul

Current Opinion in HIV & AIDS: March 2015 - Volume 10 - Issue 2 - p 123–127
doi: 10.1097/COH.0000000000000139
GENOMICS IN HIV INFECTION: Edited by Amalio Telenti
Editor's Choice

Purpose of review: This review will highlight some of the recent advances in genome engineering with applications for both clinical and basic science investigations of HIV-1.

Recent findings: Over the last year, the field of HIV cure research has seen major breakthroughs with the success of the first phase I clinical trial involving gene editing of CCR5 in patient-derived CD4+ T cells. This first human use of gene-editing technology was accomplished using zinc finger nucleases (ZFNs). Zinc finger nucleases and the advent of additional tools for genome engineering, including transcription activator-like effector nucleases (TALENS) and the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system, have made gene editing remarkably simple and affordable. Here we will discuss the different gene-editing technologies, the use of gene editing in HIV research over the past year, and potential applications of gene editing for both in-vitro and in-vivo studies.

Summary: Genome-engineering technologies have rapidly progressed over the past few years such that these systems can be easily applied in any laboratory for a variety of purposes. For HIV-1, upcoming clinical trials will determine if gene editing can provide the long-awaited functional cure. In addition, manipulation of host genomes, whether in vivo or in vitro, can facilitate development of better animal models and culture methods for studying HIV-1 transmission, pathogenesis, and virus–host interactions.

Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA

Correspondence to Paul Bates, Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. E-mail:

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The HIV field has made significant advancements since the early days of the epidemic in understanding the virus biology and developing effective therapies to inhibit replication. Today, thanks to highly-active antiretroviral therapy (HAART), patients can live with HIV for decades before AIDS progression and transmission rates are on the decline in the USA. Because of the latent reservoir of the HIV, however, HAART cessation unfailingly leads to viral outgrowth and viremia. Long-term HAART usage also has its own set of side-effects including coronary artery disease [1], osteoporosis [2], and kidney failure [3], furthering the need for curative strategies.

The story of Timothy Brown, better known as the Berlin patient, is an optimistic one for the community. Brown, suffering from acute myeloid leukemia and HIV-1 infection, required a bone marrow transplant and was given donor cells from a individual homozygous for a naturally occurring 32-bp deletion in chemokine (C-C motif) receptor 5 (CCR5), an HIV-1 coreceptor required for entry [4]. Following the success of the transplant, Brown discontinued HIV-1 therapy and had no detectable viremia for over 6 years [5]. From this observation, CCR5 modification has become a gene therapy target for curative HIV research. In 2014, Tebas et al.[6▪▪] published the first phase I clinical trial of reconstituting HIV-1 patients with autologous CD4+ T cells that had been subject to targeted CCR5 disruption using a designer zinc finger nuclease (ZFN). Although only intended to test safety of the intervention, the treatment had observable efficacy, when patient viral loads started to decrease during the HAART cessation period following the engraftment. These promising observations are driving additional clinical trails and the hope that a functional cure is in our future.

As illustrated by the ZFN–CCR5 trial, the field of genetic engineering is changing the way we think about gene therapy and treatment strategies. In a little over a decade since the completion of the Human Genome Project, the field of human genetics is again transformed by the development of tools for precise modification of genomes. Although the ZFN used in the CCR5 trial was developed by Sangamo over several years, recent advances in designer nuclease technology have greatly reduced the time required to design and test these tools. Moreover, the cost of assembling designer nucleases has also decreased, making them widely available. In this review, we will compare the various designer nucleases available including their delivery methods and applications. Furthermore, we will discuss how gene editing is currently being applied in the search for a cure and how these tools can facilitate the development of systems to better study HIV in vitro and in vivo.

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There are three types of nucleases that are currently used for genome engineering (Fig. 1). These include ZFNs, transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPRs). Although each type of nuclease recognizes its DNA targets differently, the effective outcomes are all the same. Upon substrate (DNA target sequence) recognition, the endonuclease creates a double-stranded break in the DNA that can be repaired with high fidelity by homologous recombination or more likely through the error-prone mechanism of nonhomologous end joining. Repair by nonhomologous end joining can result in small insertions and deletions (indels) surrounding the break, which can subsequently lead to frameshift mutations that disrupt proper gene expression when occurring in coding exons. These designer nucleases can also be delivered to cells or accompanied by donor DNA that can serve as a template strand to preferentially induce homologous recombination-mediated repair, leading to insertion of new sequences or seamless replacement of single-nucleotide polymorphisms (SNPs) both in vitro and in vivo[7–10].

The oldest and most well characterized are ZFNs that are composed of an N-terminal zinc finger DNA-binding domain fused to one-half of a FokI cleavage domain at the C-terminus. Two ZFNs must bind to an adjacent stretch of DNA for FokI to dimerize and become activated. Each zinc finger domain recognizes three base pairs of DNA in a modular fashion with each ZFN arm containing three to four domains, adding additional levels of context specificity. The protein–DNA interactions are empirically determined and as such, design of ZFNs can be time consuming. To reduce off-target effects, the two FokI domains have been engineered as heterodimers that can only create double-stranded breaks when the two different arms come together [11].

More recently, TALENs have functioned as a second-generation designer nuclease, whereby a modular protein arm is fused to one-half of a FokI cleavage domain. In contrast to ZFNs, TALENs are made up of ‘fingers’ that recognize a single base pair of DNA. Although these constructs are less context specific, the protein–DNA interactions are much more predicable and do not have to undergo the same level of rigorous optimization as ZFNs. In addition, since TALEN design follows a general code, groups have developed software tools to assist researchers in designing their own TALENs from commercially available assembly kits. Although the design of TALENs is generally more straightforward than ZFNs, production of these nucleases can be challenging.

The most recent and likely to become the ubiquitous form of gene editing is the CRISPR/Cas9 system. CRISPRs were initially discovered as a bacterial adaptive immune system for incoming foreign phage/plasmid DNA, but were subsequently reengineered by several groups to target DNA in eukaryotic cells [12–15]. Unlike ZFNs and TALENs, DNA-binding activity is conferred through hybridization with a guide RNA (gRNA) and follows simple Watson–Crick base pairing. This gRNA has a short (20 bp) complementary region fused to a larger RNA scaffold that is recognized and bounded by the CRISPR associated protein 9 (Cas9) endonuclease. The only stringent requirement for successful targeting is the presence of a protospacer adjacent motif (typically NGG) in the target DNA immediately downstream (3′ proximal) of the RNA-binding region. Although each of the designer nucleases are entirely customizable, ZFNs and TALENs require more time spent on design, assembly, and optimization than CRISPRs, making the CRISPR system the easiest and the cheapest gene-editing platform, as illustrated by the sheer number of articles published in the 2 years since they were first described for eukaryotic use.

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Delivery of designer nucleases in tissue culture can by accomplished through multiple routes. Standard transfection and nucleofection methods can be used for plasmid DNA delivery, whereas delivery by viral transduction is also quite successful. Viral transduction is the most efficient for nuclease delivery and generally has less toxicity issues than nucleofection. Adenoviral vectors rather than lentiviral vectors must be used with TALEN constructs because of their highly repetitive elements, which have the potential to recombine. Lentiviral vectors are well tolerated when used with CRISPRs, and numerous groups have deposited into AddGene, a nonprofit organization in Cambridge, MA, dedicated to making it easier for scientists to share plasmids, lentiviral constructs specifically for gRNA and Cas9 delivery. The method of delivery also depends on the target cell, as some cell lines are not easily transfected.

Moving in vivo, there has been great success in generating transgenic animals using CRISPRs, including modification of multiple genes within the same organism [16–20]. Delivery of gRNA and Cas9 is accomplished by microinjection of in-vitro-transcribed RNA into one-cell embryos. Because the RNA is eventually degraded, little toxicity is observed in manipulated embryos and long-term accumulation of off-target modifications (e.g., in the case of stable transduction) is mitigated. In terms of therapeutic applications, delivery of the nuclease needs to be efficient, occur at a large scale (108–109 cells), and be highly reproducible. In a CCR5–ZFN phase I trial, the ZFN was delivered by a replication-defective Ad5/35 vector [6▪▪]. Delivery by nonintegrating viruses will likely be the route of delivery in future studies.

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An exciting utilization of designer nucleases has been in curative HIV research. The CCR5–ZFN trial is promising for the field, and subsequent trials are under way by Sangamo to investigate the dosing of modified CD4+ T cells, with and without cyclophosphamide pretreatment [21–23]. Time will tell if this is a viable treatment option and whether or not HIV-infected individuals will be able to live without daily HAART.

In addition to gene therapy trials, genome engineering can be applied to better understand virus–host interactions. A nonhuman primate (NHP) model for HIV-1 infection is still lacking in the field, but two groups have shown that transgenic monkeys can be made using CRISPR/Cas9 [24▪] and TALENs [25▪]. Manipulation of NHP to remove barriers to cross-species transmission (e.g., TRIM5α, tetherin) has the potential to elicit HIV-1 susceptibility. In addition, humanized mouse models of HIV infection have been helpful for studying HIV pathogenesis in vivo. Gene editing has the ability to make these small animal models better by targeted replacement of murine genes with the human homolog (e.g., cytokines) to recapitulate the human adaptive immune system better.

Studying HIV–host interactions in vitro can also be greatly enhanced by the use of genome editing. With the ease that CRISPRs can be assembled and delivered, one can study the effects of knocking out genes of interest using the standard Cas9 nuclease or modulate gene expression with catalytically inactive Cas9 fused to transcription activators or repressors [26–28]. Similar systems are also available with TALENs [29,30]; however, the more laborious process of producing TALENs suggests that the CRISPR systems will be more commonly utilized. In addition, the Cas9 nickase [31] (which creates a single-stranded break) delivered with a donor template to promote homologous recombination can be used to recapitulate interesting SNPs or polymorphisms that may be important modulators of susceptibility/resistance to HIV infection or replication.

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The therapeutic potential of genome editing is already evident with the current trials involving patient-derived CD4+ T cells in HIV-1 infected individuals [6▪▪,21–23].

In the first reported phase 1 clinical trial, Tebas and colleagues [6▪▪] modified autologous CD4+ T cells from HAART-controlled HIV-infected individuals with ZFNs targeting CCR5. Following successful engraftment, several patients underwent a treatment interruption during which unmodified CD4+ T cells declined at a significantly faster rate than their modified counterparts, suggesting the latter had a selective advantage. Furthermore, viral loads started to decline before the end of the treatment interruption, potentially reflecting control of the virus by the host immune system.

Many studies have been done to look at the off-target effects of ZFNs, and although there is risk involved, the potential benefit is much greater. Furthermore, the use of CD4+ T cells mitigates the risk of tumor development when compared with proposed studies in which hematopoietic stem cells will be edited. In contrast with ZFNs, TALENs have not been looked at closely with regard to their off-target potential. The timing of TALENs commercial availability nearly coincided with the first publications involving CRISPR/Cas9 use in eukaryotic cells, leading most laboratories to focus on the latter. Several studies looking at CRISPR/Cas9 off-target effects have been published, although reports are somewhat conflicting. The relatively extensive use of CRISPRs in the generation of transgenic animals with seemingly normal development is encouraging for their therapeutic use.

An additional HIV-interacting host factor has been targeted for preliminary curative research. TALENs [32] and ZFNs [33] were used to generate deletions in the HIV integration co-factor lens epithelium-derived growth factor (LEDGF), also known as PS1P1, resulting in impaired integration and virus spread. Although LEDGF knockout is tolerated in mice, it is unclear if this is a feasible target in humans and if any additional benefit would be provided in combination with ZFN–CCR5 therapy. Moving away from targeting host–factor determinants for infection (e.g., CCR5), three groups have recently published a method for disrupting the HIV provirus using ZFNs, TALENs, and CRISPRs targeting the HIV long terminal repeat (LTR) [34,35,36▪]. These studies were done in vitro in HIV-transduced cell lines. Although targeting the HIV reservoir to affect an HIV cure is an interesting concept, clinical trials attempting to ‘flush out’ the reservoir using histone deacetylase (HDAC) inhibitors have been ineffectual thus far [37,38]. Using a gene therapy approach to disrupt the HIV reservoir has several obstacles to overcome in terms of identifying and successfully targeting the entirety of the reservoir, which would seem to be required for a functional cure.

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Recent advances in the generation of designer site-specific nucleases, specifically the advent of the CRISPR/Cas9 systems, have the potential to revolutionize many studies on HIV-1 biology. Immediate applications of this technology include in-vitro studies of required host genes or innate restriction systems and development of genetically altered animal models for HIV-1 infection. Longer term, it seems likely that the ease with which gene-editing systems can be developed and tested will lead to safe and efficient production of gene edited terminally differentiated or hematopoietic stem cells that are resistant to HIV-1 infection for reintroduction into patients. Less clear is the path to deployment of gene-editing technologies for destruction of the HIV-1 reservoir in patients.

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Financial support and sponsorship

M.J.D. was funded by grant T32GM007229, and P.B. was funded by NIH grants R01AI081913 and P51OD011104-52 Supplement.

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Conflicts of interest

There are no conflicts of interest.

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Papers of particular interest, published within the annual period of review, have been highlighted as:

▪ of special interest

▪▪ of outstanding interest

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CRISPR/Cas9; gene editing; HIV-1; transcription activator-like effector nuclease; zinc finger nuclease

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