Research Article: The therapeutic landscape of HIV-1 via genome editing

Date Published: July 14, 2017

Publisher: BioMed Central

Author(s): Alexander Kwarteng, Samuel Terkper Ahuno, Godwin Kwakye-Nuako.

http://doi.org/10.1186/s12981-017-0157-8

Abstract

Current treatment for HIV-1 largely relies on chemotherapy through the administration of antiretroviral drugs. While the search for anti-HIV-1 vaccine remain elusive, the use of highly active antiretroviral therapies (HAART) have been far-reaching and has changed HIV-1 into a manageable chronic infection. There is compelling evidence, including several side-effects of ARTs, suggesting that eradication of HIV-1 cannot depend solely on antiretrovirals. Gene therapy, an expanding treatment strategy, using RNA interference (RNAi) and programmable nucleases such as meganuclease, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins (CRISPR–Cas9) are transforming the therapeutic landscape of HIV-1. TALENS and ZFNS are structurally similar modular systems, which consist of a FokI endonuclease fused to custom-designed effector proteins but have been largely limited, particularly ZFNs, due to their complexity and cost of protein engineering. However, the newly developed CRISPR–Cas9 system, consists of a single guide RNA (sgRNA), which directs a Cas9 endonuclease to complementary target sites, and serves as a superior alternative to the previous protein-based systems. The techniques have been successfully applied to the development of better HIV-1 models, generation of protective mutations in endogenous/host cells, disruption of HIV-1 genomes and even reactivating latent viruses for better detection and clearance by host immune response. Here, we focus on gene editing-based HIV-1 treatment and research in addition to providing  perspectives for refining these techniques.

Partial Text

Human immunodeficiency virus-1 (HIV-1) infection is still a major contributor to global disease burden. The brunt of the infection is borne mostly by resource-limited populations [1]. Despite much effort by regional and international public health organizations, sub-Saharan Africa accounts for approximately 70% of all 36.73 million people living with HIV-1 world-wide [2]. On the other hand, the availability of early treatment therapies are changing the epidemiology of the disease, contributing to decreasing HIV-1 incidence as a result of drastic reductions of the risk of transmission of the infection [1].

The call to end the global pandemic might be achieved through two therapeutic approaches. Firstly, the sterilizing approach, which theoretically implies purging all HIV-1 latent reservoirs (as described above). Secondly, the functional cure approach, which seeks to empower the host immune defences to fight the infection and other opportunistic infections that arise as the disease progresses. Generally, both approaches are effective and heavily depend on HAART. Research into anti-HIV-1 gene therapy has intensified following the so called “Berlin-patient” where scientists eradicated HIV from his body after receiving a bone marrow transplant. Therefore, gene and nucleic acid based therapies including gene editing with programmable nucleases, RNA decoy, DNA/RNA aptamers, ribozymes, antisense, inhibitory proteins, fusion inhibitors and sh/siRNA have also been developed of which some are candidates for ongoing clinical trials (Table 1) [39–48].Table 1Anti-HIV-1 gene therapy landscapeTherapeutic approachGene therapyInterventionsTargetsIdentifierStage/statusCompany/InstituteDrug shRNA peptideDual anti-HIV gene Transfer construct, LVsh5/C46 (Cal)CCR5 shRNA; C46 peptide BusulfanHost co-receptor; viral Env; transplant conditioningNCT01734850Phase I/II recruitingCalimmune/CalTech/UCLAshRNALong term follow up of delayed adverse events in Cal-1 recipientsBlood test for general health, complete blood count and Cal-1 specific analysesNCT02390297Recruiting by invitationCalimmune/CalTech/UCLAEndoribonucleaseRedirected MazF-CD4 autologous T-cellsCCR5 MazFHost co-receptorNCT01787994Phase I ongoingUniversity of PennsylvaniaRibozymeAutologous CD34+ HSCs transduced with anti-HIV-1 ribozyme (OZ1)Tat-vpr anti-HIV ribozymetat-vpr mRNANCT00074997Phase II completedJanssen-Cilag Pty Ltd, UCLARibozymeLong term follow up of OZ1 Gene therapyBlood test for quantitative marking of the gene transfer product in PBMCs overtimeNCT01177059Phase II Recruiting by invitationJanssen-Cilag Pty Ltd, UCLAAntisenseTolerability and therapeutic effects of repeated doses of autologous T cells with VRX496VRX496 antisense RNAEnv mRNANCT00295477Phase I/II Ongoing/ not recruitingUniversity of Pennslyvernia/NIAIDAntisenseSafety and efficacy of T-cell genetic immunotherapyVRX496 antisense RNAEnv mRNANCT00131560Phase II OngoingVIRxSYS CorporationRibozymeL-TR/Tat-neo in HIV+ Patients with Non-Hodgkin’s LymphomaTat ribozymeTat-rev mRNANCT00002221Phase II completedRibozyome/City of HopeInhibitory peptidesM87o autologous HSCs for HIV+ patients with malignant diseaseC46 peptideViral EnvNCT00858793Phase I/II suspendedUniversity Medical Center Hamburg-EppendorfRibozyme, inhibitory peptideC46/CCR5/P140K modified autologous HSCs in patients with lymphomaC46 peptide, CCR5 ribozyme, MGMTP140 K mutantViral Env, CCR5 mRNA, Alkylating agent resistanceNCT02343666Phase I, recruitingFred Hutchinson Cancer Research Center/NCI/NHLBIshRNA,peptideAutologous transplantation of HSCs with LVsh5/C46 (Cal-1) for treatment of HIV-related lymphomaCCR5 shRNA, C46 peptideHost co-receptor, viral EnvNCT02378922Phase I recruitingFred Hutchinson Cancer Research Center/NCIshRNA, ribozyme,RNA decoy, drugsrHIV7-shI-TAR-CCR5RZ-transduced HSC in patient with AIDS-related Non-Hodgkin Lymphomatat/rev shRNA, TAR decoy, CCR5 ribozyme, Prednisone, Rituximab, Etoposide, Doxorubicin Hydrochloride, Vincristine Sulfate, CyclophosphamideViral mRNA, Viral tat proteinm, CCR5 mRNA, Transplant conditioningNCT02337985Pilot recruitingCity of Hope Medical Center/NCIshRNA, ribozyme,RNA decoy, drugsrHIV7-shI-TAR-CCR5RZ-transduced HSC in patient with AIDS-related Non-Hodgkin Lymphomatat/rev shRNA, TAR decoy, CCR5 ribozyme, BusulfanViral mRNA, Viral tat proteinm, CCR5 mRNANCT01961063Pilot recruitingCity of Hope Medical Center/NCIshRNA, ribozyme,RNA decoy, drugsrHIV7-shI-TAR-CCR5RZ-transduced HSC in patient undergoing stem cell transplant for AIDS-related lymphomatat/rev shRNA, TAR decoy, CCR5 ribozyme, carmustine, cyclophosphamide, etoposideviral mRNA, viral tat protein, CCR5 mRNA, transplant conditioningNCT00569985Pilot ongoing, not recruitingCity of Hope Medical Center/NCIshRNA, RNA decoyshRNA/TRIM5alpha/TAR decoy-transduced Autologous HSC in patient with HIV-Related LymphomaCCR5 shRNA, RNF88,TAR decoyHost co-receptor, Gag p24, Viral tat proteinNCT02797470Phase I/IICity of Hope Medical Center/NCIRNA decoyRNA decoy (ex vivo retroviral modified CD34+ HPC)Rev reponse element decoyRev ProteinNCT00001535Phase 0-pilotChildren’s HospitalRedirected high affinity gag-specific autologous T cellsWT-gag-TCR or alpha/6-gag-TCRCD8 TCRNCT00991224Phase I completedUniversity of Pennslyvernia/AdaptimmuneZFNT-cells modified at CCR5 gene by ZFN SB-728mRCCR5 ZFNCCR5 DNANCT02388594Phase 1 recruitingUniversity of Pennsylvania/NIAIDRepeated doses of SB-728mR-T after cyclophosphamide conditioning in HIV+ on HAARTCCR5 ZFN (SB-728mR-T)CCR5 DNANCT02225665PHASE I/II ongoingSangamo biosciencesAutologous T cells modified at CCR5 gene by ZFN SB-728CCR5 ZFNCCR5 DNANCT00842634Phase 1 completedSangamo biosciences/ Uni. Of PennsylvaniaAutologous T-cells genetically modified at the CCR5 gene by zinc finger nucleases (SB-728-T) in HIV-infected patientsCCR5 ZFNCCR5 DNANCT01044654Phase I completedSangamo TherapeuticsSafety of ZFN CCR5-modified HPS/progenitor cells in HIV+SB-728mR-HSPC infusion after busulfan conditioningCCR5 DNANCT02500849Phase 1City of Hope Medical Center|Sangamo TherapeuticsAutologous T-cells genetically modified at the CCR5 Gene by zinc finger nucleases in HIV-infected subjectsCCR5 ZFN (SB-728-T)CCR5 DNANCT01252641Phase I/II completedSangamo Therapeutics

Treatment with HAART, the primary treatment strategy, has greatly impacted the epidemiology of HIV-1 infection changing the previously life-threatening disease into a chronic disease. The move by world leaders to make these drugs available to endemic regions, particularly developing nations, led to significant reduction in the number of AIDS-related deaths as well as an increase in the quality of life of infected individuals [49, 50]. However, HAART is intensive and life-long, usually leading to treatment fatigue, with considerable side effects [50]. Moreover, poor pharmacokinetics of these drugs and tissue toxicity, on top of viral resistance after prolonged treatment, have also been widely documented throughout the volumes of scientific literature and clinical practice [51]. Added to these are the huge economic and logistical challenges borne by developing countries in order to make treatment sustainable. The constellation of drawbacks warrants the development of robust and effective treatment regimens to supplant HAART, which will result in better treatment and management as well as the possible eradication of the virus. The advancement of biomedical research and engineering, nano-delivery of drugs to specific and key anatomical barriers hold the promise of increasing the efficiency of HIV-1 chemotherapy [52–54].

Anti-HIV-1 nanomedicine involves the administration of minute (on the nano-scale, 10−9 m) anti-HIV-1 therapeutic agents to allow precise delivery to virtually any therapeutic target sites particularly HIV-1 sanctuary sites such as the central nervous system. The development of biocompatible, biodegradable, non-toxic nanoparticles are feasible depending on the material of manufacturing and are therefore a keen research focus in biomedical engineering [55]. Nanotechnology-based anti-HIV-1 therapeutic agents could range from various drug formulations to gene therapy toolkits (such as RNA interference and anti-HIV-1 ribozymes), which could either be bound to or encapsulated in nano-carriers [56]. Anti-HIV-1 nano-based therapeutic agents have been upheld for their ability to facilitate stable and prolonged drug circulation coupled with the ability to specifically target intended cells/tissues with improved toxicity profiles and low side effects [53]. These ground-breaking techniques facilitate the permeation of the blood–brain barrier of the CNS with remarkable precision and accuracy [57]. However, a move to translate nanotechnology-based anti-HIV-1 agents into clinical practice would require a critical review of existing delivery routes as well as the development of novel delivery routes for nano-formulations. This precaution has now become necessary given that both the pharmacodynamics and pharmacokinetics (absorption, distribution, metabolism and elimination) of nano-formulations are at times affected by the mode of delivery [58]. Such assessments would deepen our knowledge of the efficacy and safety of this treatment approach while opening up new frontiers for HIV-1 research and treatment. A critical assessment of the cost-to-benefit ratio is of equal importance, particularly to ensure wider coverage of middle and lower income individuals as well as resource-limited countries in the upcoming years.

RNA interference is a widely used technique in endogenous cells and biomedical research for regulating gene expression and cellular defense against viral infections [59]. The technique has served as the bedrock for the elucidation of complex signaling pathways in biological systems, functional genomics and gene therapy. RNA interference has been extensively applied to the elucidation of HIV-1 pathogenesis as well as identification of novel therapeutic targets for controlling HIV-1 infections.

Conversely, despite the popularity of RNAi mediated anti-HIV-1 treatment and research, some trade-offs exist. Of particular importance, is the high likelihood of generating viral escape mutants also known as siRNA escape mutants. This is due to the high error rate of the viral reverse transcriptase (1 in 1000 nucleotides per replication cycle) hence a change in even 1 bp could lead to mutations in the targeted sequences therefore limiting the regulatory effects of siRNAs [69]. However, one of the surest ways of offsetting this challenge is by deploying multiple anti-HIV-1 siRNAs. Another is the combination of other anti-HIV-1 therapeutics, in particular antiretroviral drugs.

The arrival of gene editing tools such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins (CRISPR–Cas9) has revolutionized biomedical research. Similar to other seminal scientific breakthroughs, the adoption of these reagents by modern research have increased exponentially since their invention—and undoubtedly proves promising as arsenals for eradicating HIV-1 infections as well as other viral infections (such as Hepatitis B and C virus, human papilloma virus and herpes simplex virus) even in resource-limited populations.

The structure of ZFNs is basically the combination of 2 domains: the nonspecific FokI restriction endonuclease for cleavage of targeted sequences and the custom-designed Cys2-His2 zinc-finger proteins (ZFPs) for specific DNA-binding (Fig. 1). The domains are stabilized by zinc ions. Unlike other DNA binding proteins, which depends on the twofold symmetry of the double helix, ZFNs have the advantage of being linked linearly in tandem to recognize nucleic acids of varying lengths, allowing unprecedented combinatorial possibilities for specific gene targeting and manipulations [70]. Ideally, ZFN subunits recognize target sequences in a head-to-tail conformation. After recognition and binding of ZFNs to specific genomic loci, there is the dimerization of the two nuclease domains which leads to a double-stranded break (DSB) of the targeted DNA [70].Fig. 1Zinc finger nucleases

TALENs are structurally similar to ZFNs comprising of a TALE DNA-binding region and a FokI restriction endonuclease domain (Fig. 2) [73]. Transcription activator-like effectors (TALEs) are naturally occurring DNA binding proteins from the plant bacterial pathogen, Xanthomonas [74]. In contrast to ZFNs where each finger module recognizes three target DNA nucleotides, TALE proteins contain a highly conserved, central domain, usually consisting of 33–35 amino acid TALE repeats for which each protein monomer is capable of recognizing single base pairs of the target DNA [75]. However, the specificity of these DNA-protein interactions are dictated by two hypervariable residues as shown by studies, which investigated the crystal structure of TALEs bound to DNA. The results from these studies show that each TALEN repeat forms a two-helix structure connected by a loop which presents the hypervariable residues into the major groove as the protein wraps around the DNA in a super-helical structure [76, 77]. The flexibility of joining these modular TALE repeats to form long arrays with custom DNA-binding specificities has proved useful in targeted gene editing of a variety of cells for therapeutic purposes [78–80]. Although TALENs are cost effective when compared to ZFNs, they are difficult to generate. The bulkiness of both ZFNs and TALENs makes it more difficult to deliver these reagents to several targeted cell types [81]. It has been shown that the presence of multiple sequence repeats in TALEN genes renders them unsuitable cargos for lentiviral vector repeats [81]. However, the single-nucleotide precision give rise to superior editing efficiency with minimal off-target and cytotoxicity effects when compared to ZFNs thereby making TALENs good candidates for sequence-specific genome modification [82].Fig. 2TALENS

The emergence of CRISPR–Cas9, a proof-of-principle technique based on the adaptive immune systems of bacteria and archae [108] has transformed the therapeutic landscape of HIV-1. CRISPR–Cas9, has gained so much popularity in the research community due to the preciseness, cost-effectiveness and simplicity with its design thus allowing superior genetic manipulations of targeted sequences [109]. Unlike the former designer nucleases (ZFN and TALENs) CRISPR–Cas9 uses a specially designed guide RNA (gRNA) to direct a nuclease (Cas9) to specific genomic loci for genomic modification (Fig. 3) [110]. The disrupted genomic DNA is then repaired either by non-homologous end joining (NHEJ) or homologous recombination (HR) (Fig. 4). DNA repair via non-homologous end joining usually leads to mutations that interrupt the open reading frame, which could lead to gene inactivation when a template is provided.Fig. 3CRISPR–Cas9Fig. 4DNA repair mechanism

Another valuable addition to the gene editing toolbox has been NgAgo, a DNA-guided endonuclease [113] with possibilities of generating site-specific modification of human cells. Unlike CRISPR–Cas9, NgAgo-gDNA system operates without a protospacer-adjacent motif (PAM) with remarkable low tolerance to guide-target mismatches as well as high efficiency in editing (G+C)-rich genomic loci. Although promising, the reproducibility of the original protocols could prolong the huge benefits such a tool could offer biomedical researchers with profound interest in gene modifications [114]. There are no reported studies using this latest addition to the genome editing toolbox for HIV-1 treatment and research, suggesting the need for future research to explore the potentials of NgAgo for HIV-1 research.

In this review, we have highlighted some of the mechanisms underlying the application of genome editing as a major control to augment existing strategies against HIV-1 infection. It will be interesting to investigate the efficacy of clearing HIV-1 proviral DNA while shedding more light on the outcome of such strategies on the health and safety of individuals.

 

Source:

http://doi.org/10.1186/s12981-017-0157-8

 

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