Introduction

It is demonstratedthata cc-chemokine receptor (CCR)-based targeted therapy canplaycritical role in more effective cure of patients infected by human immune-deficiency virus-1 (HIV-1) [1-4]. CCRs are a group ofG-protein coupled family of receptors(GPCR) andapproximately 800 genes have beenintroduced that encoded functional GPCR and made up about %1 of human genome[5, 6].It is demonstrated that about half of clinical drugs designCCR5 as a Novel Cell and Gene Therapy for GPCRs, with blocking their ligands or increasing ligands accessibility. Sotheir rolesin our bodiesare very important[7].

Human GPCRs consist of six families whose major groups are A,B and C andtheir largest group is A family. One of the most popular subfamilies of class A is CCR5 which for many years hasattracted scientists. The vast majority of investigations have focused to provide the novel approaches to the treatment of HIV based on targeted therapy. We have previously suggested induction of resistance to HIV by CCR5 gene therapy and stem cell transplantation [8, 9]. Here we present most recently discovered approaches to the treatment of HIV, based on CCR5, which may define the better treatment, especially target therapy.

CCR5 genetic basis, structure and molecular signaling

CCR5 is encoded by CMKBR5 gene located on p21.31 region of human chromosome 3[10].Protein productof this gene includes 3 sections[11, 12]: 1) A seven helical trans-membrane domain which provide 3 extra-cellular and 3 intra-cellular hydrophilic loops. 2) C-terminal residue that regulates receptor by serine-threonine phosphorylation. 3) N-terminal residue that is bound to ligand. MIP 1α,β (also knownas CCL3, CCL4), RANTES (also known as CCL5) and the other β-chemokines areligands that are bound to NT site in CCR5 [12, 13]. After ligand binding to NT or 3 extracellular loops, a molecular mechanism activate that cause receptor conformation change and the dissociation of G-protein subunits. Gα-GTP and Gβγ subunits are freed and regulate enzymesactivity such as adenylate cyclase, phospholipase C isoforms and ion channels. These enzymes regulate protein kinase function, finally increase intracellular ca²⁺ ions and stimulate chemotaxis[14].Pharmacologist have named GPCR as “spare receptor” because full biologic response happen after 5%occupancy [15]. Thus GPCRs associatewithcell metabolism, growth, migration, differentiation and death of multiple cell type (apoptosis)[16, 17].

Resistance to HIV-1 infection byCCR5

Any change in the sequence of gene variants causes alternation in HIV infection or AIDS process, named as AIDS Restricted Gene (ARG). Some of these variants are detected in special group population which causes natural selection and/or genetic drift different ARG polymorphism/mutation. To this date, more than 35 ARGs have been identified. First of them is CCR5Δ32 and [18-22].This32bp deletion(CCR5Δ32) in the single-coding exon provides resistance mechanism to HIV/AIDS infection[12, 23].CCR5Δ32 is caused by replicative slippage by RNA pol due to the presence of a direct repeat flanking the deleted region which leads to elimination of second extracellular loop on receptor. Therefore, HIV-1 enters to bloodstream but could not infect CD4⁺ T cell and macrophage[12]. A number of studieshave shown thatindividual with homozygote CCR5Δ32allele exhibits a natural resistance to HIV and Acquired immunodeficiency syndrome(AIDS)[12, 23, 24].It is also worth mentioning that heterozygote individual after exposing to human immunodeficiency virus display slower progression to AIDSthan homozygote individualfor the wild type allele [25, 26].In Northern Europe, Caucasian population indicate the highestCCR5Δ32, 20% of whom are heterozygote and 1% homozygote for this mutation, respectively [27]. Because of these remarkable detections, CCR5 isknown as a major co-receptor, even when immune-cells presentCD4 on their surface.If CCR5 isnot presentedon cell surface (following knock-out it or when using its inhibitors) HIV-1couldnot contaminate cells [28, 29].

CCR5Δ32is highly resistant to HIV-1 infection, but not completely. SinceCXCR4is another co-receptor for HIV-1 entries. So CCR5 is not the only co-receptor for HIV-1 infection and one of the reasons that HIV/AIDS have epidemiological heterogeneity is genetic variants in host receptor and co-receptors (CCR5 or CXCR4) that HIV can switch itself to per variant[30].

resistance to HIV-1 infection or delay to AIDSprocess byAnother polymorphisms

In addition to CCR5Δ32,Some other mutations/polymorphisms have been discoveredwhichprovidethe resistance mechanism, suchasCCR5/m303 (table 1)[31], IDH1C[32]. But although some ofthe otherslikeCCR5Δ32 heterozygote are susceptible to HIV infection,theydelay the AIDS progression,such as CCR2 64I[33], SDF1 3‘A[25], HLA B*57[18], HLA B27[34], KIR 3DS1[35, 36], PROX1 Hap-CGT[37], ACSM4 A[38].

Thus, the variant of host genotype may change the function or alternate gene expression after HIV-1 contamination, known as HIV-dependency factors (HDFs). HDFs are needed for HIV-1 infection process, transmission, viral loading and challenge with immune-system[39]. However, host genetic background is a part of factors that affect HIV-1infection. The others are HIV acquisition, immune-system condition and HAART (highly-active-anti-retroviral therapy)results.

Current therapeutic methods: benefits and limitations

To date, several drugs have been introduced for inhibition of AIDS progression, like enfuviritude (T20) and maraviroc. Maravirocisa co-receptor antagonistblocks interaction between CCR5 and envelope (env) proteincoating HIV-1 surface[40].Enfuviritudesact asfusion inhibitordisrupting conformation change in glycoprotein-41 (gp41)[40]. In addition to,highly active anti-retroviral therapy (HAART)is emerging which targets virus enzymes (Table 2). It isshowed that ARThave significantpositive effect on the reduction of latent reservoir virus in immune system atan early stage or immediately after informing of HIV-1 infection. For example,when an infant was burn from a mother that infected by HIV-1,after 30 hours of birth undergoneART and continued until 18 months. In the months30, no pro-viral DNA or plasma RNA of HIV was detected in peripheral mononuclear cells[41].

One of the problems in HIV-1 treatment by ART is “drugs resistance”. This phenomenon mainly caused by new mutation patternsin HIV-1 genes that virus need for its essential proteins like protease[42]. Hence,HAART could not eradicate virus, although reduce its replication significance. In addition, theyshouldbe used long timeand maybe discontinueof themin any time causeRNA and DNA virusrebounding from latent reservoirs. Moreover, HAART cannot act in different individuals by the same efficiency. High cost and some side effects following long-term therapy are another restrictionthe use of HAART[43, 44].

Recent treatment strategies are focused on gene therapy especially against HIV and other refractory disease, we present some study in this field previously[8, 9, 45-49]. Here, we present most recently discovered approaches to the treatment of HIV, based on CCR5, which may define the better treatment, especially target therapy.

Novel therapeutics approaches:stem cell transplantation and gene-modification

Novel treatment strategies with stem cell transplantation (SCT) overcome to some problems that observed in ART[8, 9].Successful allogeneic SCT haveperformed for acute myeloid leukemia (AML) in a patient co-infected by HIV and HCV andhad been undergone HAART in 2002[50]. Following this, Hutteret al. in 2009 transplanted allogeneic (CCR5 Δ32/Δ32) stem cell as a treatment for “Berlin patient”whowas suffering from AML and HIV infection[1]. The patient was undergoing HAART for 10 years and discontinued them after SCT[1].Rebounding of HIV may beobserved if HAART discontinued because of another CXCR4 co-receptorexistence[51], but in this patient after 20 months not observed signs of virus activating or replicating [1].Hutteret al. sequenced CXCR4 patient and checked its variants, but not detected any trail of CXCR4 and concluded HIV-1 in this patient was not binding with CXCR4 as a co-receptor. Number of CD 4⁺ T-cells after HSCT for CCR5 Δ32/Δ32 increased such as normal range in health people and HIV DNA or RNA was undetectable gradually [1]. Result of this treatment showedno existence of RNA or DNA virusafter 3.5 years, even when HAART had been discontinued[2, 52].Likewise, it was reported Berlin patient body remained free of HIV-1 after 5.5 years[53].

In 2012,Henrichet al. performed HSCT for two patients, Boston patients,who have heterozygote genotype forCCR5 (CCR5 WT/CCR5Δ32) and transplanted with WT CCR5 cells. Result of this study suggested that replication of DNA or RNA virus had been suppressed and HIV-1 reservoir reducedafter transplantation [3].

Although results of Hutteret al. and Henrichet al. made a revolution in HIV-1 treatment, but there were problems such as low frequency of homozygote CCR5Δ32 in population and founding a suitable HLA match donor with target patient. These problems encouraged Duarteet al. in 2015 to transplanted hematopoietic stem cell of umbilical cord blood (CB) to a patient infected by HIV-1 from CCR5 ^-/- donor that have AML [54-56]. HCT from CB not needed to stringent HLA matching like as HSCT with bone marrow. This study showed peripheral mononuclear cells were resistance to HIV-1 infection.

As describe above, artificial disruption methodswith “CCR5gene modification”were exerted widely and eliminated some problems of allogeneic HSCT[57]. Gene modification is permanent, inheritable and transmitted HIV-1 resistant cells to next generation.

For example for site-specific nuclease,zinc finger protein (ZFP) surveyed extensive. ZFP is a transcription factor that binding to Fok I restriction enzyme domains with their zinc finger motifs and provide zinc finger nuclease (ZFN)[58, 59].Theoretically, following DSB in DNA by Fok I, repair DNA mechanism applied error prone non-homologous end joining (NHEJ) [60] or homology directly repair by homologous recombination (HR) [61]. Then, small nucleotide deletion or addition observed and result in disruption of reading frame and gene expression. It could engineer with nucleases artificially. Predominant repair with ZFN is error-prone NHEJ.

First in 2005, alternation ofCCR5 with ZFN in in vitro conditionwas showed [62]. ThenCCR5 disruption by ZFN in mouse model was applied and exhibited specify and sufficient in vivofunctions for induction of resistance to HIV[63].In this manner, genetic modification with ZFNs utilized in various living organism and cell lines;Including primary T cell, HSC and humanize mice[64-67].Afterwards, CCR5 was modified by ZFN with adenovirus vector on ex vivo condition and performance wasefficiently in healthy and HIV infected CD4⁺ T cellin 2013[68].Knock out ofCCR5 by ZFP modification artificiallyand infusion of autologous CD4⁺ T cell transplantation showed safety and immune reconstitution with increasing in CD4⁺T cells,In 2014[4]. In this studyDNA virus level decreased in most patients and RNA virus level were undetectable in one patient andmodified T cells for CCR5were stable[4].

Another kind of site-specific nuclease is TALEN (Transcription Activator-like Effectors Nuclease). In comparison toZFN, TALEN has lower cytotoxicity and reduce off-target activityin CCR5 locus. But both of them could disrupt genes about 45%[69].In comparison toZFN more delivery problems observed, due to largeTALEN protein size. But had been showed that TALEN expressed by adenoviral vector [70].

In addition to ZFN andTALEN, CRISPR/Cas9 is another targeted gene disruption in HIV therapy.Clustered regularly interspaced palindromic repeats (CRISPRs) are short direct repeat (21-47 nt) with vary intervening spacer sequence that surround by CRISPR associated gene (Cas9) in bacteria. When CRISPR istranscribed, pre-crRNA converts to crRNA byRNase III and associate with trans-acting RNA (tracRNA) that diagnose target DNA.Then, this RNA duplex bind to Cas9 and the crRNA guide complex to target DNA that is complementary to spacer sequence. This ribonucleotide complex cleave target DNA with Cas9 and cause DSB[71].Some studies suggested CRISPR/Cas9 could efficientlyablate the viral genome from latently HIV-1 infected cells [72, 73]. CCR5 in primary human CD4⁺ T cells and CD34⁺ hematopoietic is targeted progenitor and stem cells and demonstrated ablate viral genes with minimal off-target mutagenesis by CRISPR/Cas9,In 2014[74].

T cell recovery and suppression of HIV-1 are achieved by gene modification and/or cell therapy. Recent approaches provide effective cure of HIV-1 but there are some challenges in CCR5 candidate for HIV treatment and need to more investigations.

Table 1: CCR5 polymorphisms that affected HIV-1infection

*CCR5Δ32: 32bp deletion in exon 1 CCR5 gene that cause second extracellular loop be defected and truncate protein created.

**CCR5 m303 (C101X): Transversion of T to A in 303 nucleotide open reading frame that created nonsense mutation in 101 amino-acid at first extracellular loop CCR5.

Table 2: Some of the current drugs for HIV treatment

Conclusion

Results of many studies have shown that CCR5 can be a therapeutic target for treatment of HIV-1.A new horizon of stem cell therapy (such as cord blood stem cell) is shifted to obtain more effective and easier methods that can apply for many people with no HLA-matching problems and donor finding restrictions. We suggest identification of another unknown polymorphism to apply more effective treatment of HIVby find out more genetic factors that promote or restrict HIV replication. We think also it would be interesting to study the therapeutic effect of autologous embryonic stem cell transplantation that modify by human artificial chromosome (HAC) and ZFN gene. It can cleavages CCR5 gene specifically and induces the resistance to HIV-1 infection.

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