CCR 5 as a Novel Cell and Gene Therapy Strategies Based on Induction of Resistance to HIV

Cc-chemokine receptor-5 (CCR5) is known as a main co-receptor in human immunodeficiency virus-1 (HIV-1) infection. So, it could be a target for inhibition of HIV1 entry into CD 4+ immune cells. Many studies showed homozygote individual with 32bp deletion in CCR5 gene had nature resistance to HIV-1. In this manner, recent treatments are focused on inducing this resistance to HIV-1 infected patients with CCR5. Berlin and Boston patients transplanted with allogeneic hematopoietic stem cell (HSC) and demonstrated effective cure for HIV-1 infection. In addition, zinc finger nuclease (ZFN) eliminated some problems of Berlin and Boston patients by site-specific CCR5 gene modification. These recent strategies declined highly-active anti-retroviral therapy (HAART) restrictions such as toxicity, low safety, the side effects following long-term consuming and virus reloading immediately after cut the drugs off. In this review, in addition of introductory biologic and immune-genetic roles of CCR5, we consider novel treatment strategies for HIV-1 infected patient by CCR5 gene targeted therapy.

It is demonstrated that a cc-chemokine receptor (CCR)-based targeted therapy can play critical role in more effective cure of patients infected by human immune-deficiency virus-1 (HIV-1) [1][2][3][4] .CCRs are a group of G-protein coupled family of receptors (GPCR) and approximately 800 genes have been introduced that encoded functional GPCR and made up about %1 of human genome 5,6 .It is demonstrated that about half of clinical drugs design for GPCRs, with blocking their ligands or increasing ligands accessibility.So their roles in our bodies are very important 7 .
Human GPCRs consist of six families whose major groups are A, B and C and their largest group is A family.One of the most popular subfamilies of class A is CCR5 which for many years has attracted 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 product of this gene includes 3 sections 11,12 : 1) A seven helical trans-membrane domain which provide 3 extra-cellular and 3 intracellular 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 known as CCL3, CCL4), RANTES (also known as CCL5) and the other β-chemokines are ligands 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 enzymes activity such as adenylate cyclase, phospholipase C isoforms and ion channels.These enzymes regulate protein kinase function, finally increase intracellular ca 2+ ions and stimulate chemotaxis 14 .Pharmacologist have named GPCR as "spare receptor" because full biologic response happen after 5% occupancy 15 .Thus GPCRs associate with cell metabolism, growth, migration, differentiation and death of multiple cell type (apoptosis) 16,17 .

Resistance to HIV-1 infection by CCR5
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 CCR5 Δ32 and [18][19][20][21][22] .This 32bp 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 studies have shown that individual with homozygote CCR5Δ32 allele 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 AIDS than homozygote individual for the wild type allele 25,26 .In Northern Europe, Caucasian population indicate the highest CCR5Δ32, 20% of whom are heterozygote and 1% homozygote for this mutation, respectively 27 .
Because of these remarkable detections, CCR5 is known as a major co-receptor, even when immunecells present CD4 on their surface.If CCR5 is not presented on cell surface (following knock-out it or when using its inhibitors) HIV-1could not contaminate cells 28,29 .CCR5Δ32 is highly resistant to HIV-1 infection, but not completely.Since CXCR4 is 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 .
Thus, the variant of host genotype may change the function or alternate gene expression after HIV-1 contamination, known as HIVdependency 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.Maraviroc is a co-receptor antagonist blocks interaction between CCR5 and envelope (env) protein coating HIV-1 surface 40 .Enfuviritudes act as fusion inhibitor disrupting conformation change in glycoprotein-41 (gp41) 40 .In addition to, highly active antiretroviral therapy (HAART) is emerging which targets virus enzymes (Table 2).It is showed that ART have significant positive effect on the reduction of latent reservoir virus in immune system at an 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 undergone ART and continued until 18 months.In the months 30, 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 patterns in 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, they should be used long time and maybe discontinue of them in any time cause RNA and DNA virus rebounding 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 restriction the 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][46][47][48][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 have performed for acute myeloid leukemia (AML) in a patient co-infected by HIV and HCV and had been undergone HAART in 2002 50 .Following this, Hutter et al. in 2009 transplanted allogeneic (CCR5 Δ32/Δ32) stem cell as a treatment for "Berlin patient" who was 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 be observed if HAART discontinued because of another CXCR4 coreceptor existence 51 , but in this patient after 20 months not observed signs of virus activating or replicating 1 .Hutter et 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 showed no existence of RNA or DNA virus after 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, Henrich et al. performed HSCT for two patients, Boston patients, who have heterozygote genotype for CCR5 (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 reduced after transplantation 3 .
Although results of Hutter et al. and Henrich et 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 Duarte et 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][55][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 methods with "CCR5 gene 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 nonhomologous 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 of CCR5 with ZFN in in vitro condition was showed 62 .Then CCR5 disruption by ZFN in mouse model was applied and exhibited specify and sufficient in vivo functions 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][65][66][67] .Afterwards, CCR5 was modified by ZFN with adenovirus vector on ex vivo condition and performance was efficiently in healthy and HIV infected CD4 + T cell in 2013 68 .Knock out of CCR5 by ZFP modification artificially and infusion of autologous CD4 + T cell transplantation showed safety and immune reconstitution with increasing in CD4 + T cells, In 2014 4 .In this study DNA virus level decreased in most patients and RNA virus level were undetectable in one patient and modified T cells for CCR5 were stable 4 .
Another kind of site-specific nuclease is TALEN (Transcription Activator-like Effectors Nuclease).In comparison to ZFN, TALEN has lower cytotoxicity and reduce off-target activity in CCR5 locus.But both of them could disrupt genes about 45% 69 .In comparison to ZFN more delivery problems observed, due to large TALEN protein size.But had been showed that TALEN expressed by adenoviral vector 70 .
In addition to ZFN and TALEN, 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 is transcribed, pre-crRNA converts to crRNA by RNase 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 efficiently ablate 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.

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 HIV by 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.

Table 2 .
Some of the current drugs for HIV treatment