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Perez, E. E. ''et al.'' [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/pdf/nihms-177273.pdf Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases]. Nat Biotechnol, 26, 808-16 (2008).


==Introduction== 
In the past decade, the field of gene therapy is progressing swiftly even though at the beginning did not show any prospects. Nowadays, there are more than 2000 different gene therapies that have entered clinical trials. Gene therapy is a method in which scientists use nucleic acid polymers to treat or prevent certain diseases by altering the patient’s genetic material. The two approaches that are commonly used include transfer of a working gene to replace the demaged one or disrupting the genes that were not working properly. In both cases, the working gene as well as genes that edit chromosomal DNA, have to be administered to the patient, to get into the demaged cell, enter the cell and express proteins to achieve desired effect. Therapeutic genes can be incorporated into a viral vector and administered to the patient as vaccine or the target cells can be transfected ''ex vivo'' and then returned to the patient, in so called adoptive cell transfer. The role of viral vectors is to deliver and incorporate DNA to the cell genome that would hopefully result in expression of proteins, which will modify cells’ phenotype back to normal. However, certain disadvantages that are present when using viral methods are circumvented with the usage of non-viral methods such as electroporation, the injection of naked DNA and the use of dendrimer, etc. Generally, the most often used method in gene therapies is the incorporation of additional gene to the cell genome. However, with the gained knowledge of the function of nucleases scientists started to use them as a reagents in editing cromosomal DNA. For example, zinc finger nucleases have become useful reagents for gene modifications of many plants and animals. It works in two steps: 
*Recognition of the sequence,
*cleveage of the chromosomal DNA.
This method allows scientists to disable or edit a demaged allele by taking advantage of the endogenous DNA repair mechanism.

==Zinc finger nuclease==
Zinc finger nucleases (ZFN) are emerging reagents for altering genes by two possible mechanisms. Both of them are based on the cell’s own DNA repair machinery. ZFNs initate a double-strand break (DSB) at specific site chosen for modification. The cells’ DNA repair machinery responds to DSB in one of two ways: homologous recombination (HR) or non-homologous end joining (NHEJ). HR occurs if there is present exogeneous template of the normal copy of the gene, while NHEJ occurs as a respond on DSB where there is no homologues gene in proximity and usually results in deletions or insertions of some of the nucleotides at the DSB site. The first mechanism may be used for correction of the demaged gene and the second one may be used for disruption of certain gene. <ref name="ref1">MILLER, J. C., HOLMES, M. C., WANG, J., GUSCHIN, D. Y., LEE, Y. L., RUPNIEWSKI, I., BEAUSEJOUR, C. M., WAITE, A. J., WANG, N. S., KIM, K. A., GREGORY, P. D., PABO, C. O. & REBAR, E. J. 2007. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol, 25, 778-85.</ref>

ZFNs may be applicable in various fields such as crop engineering, therapeutic gene correction and cell line costumization for biologics production.

ZFNs are comprised of two domains and the linker between them. Zinc finger domain is important for recognition of chosen sequence and it is coupled with cleavage domain, which is nonspecific DNA cleavage domain of the type IIS restriction enzyme, FokI. Nucleases have to dimerize before the cleavage can proceed. Overall, scientists have to engineer two ZFNs with site-specific zinc finger domains and the linker of optimized length that will link cleavage domains so that dimerization will create DSB at desired place.<ref name="ref2">URNOV, F. D., MILLER, J. C., LEE, Y. L., BEAUSEJOUR, C. M., ROCK, J. M., AUGUSTUS, S., JAMIESON, A. C., PORTEUS, M. H., GREGORY, P. D. & HOLMES, M. C. 2005. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature, 435, 646-51.</ref>

===Zinc finger domain===
Zinc finger domains in a dimer of ZFN recognize 18 or 24 bp long target sequence, depending on the number of zinc finger motifs in each domain. The hallmark of a zinc finger motif is the presence of two cysteines and two histidines, which coordinate Zn ion. It contains approximately 30 amino acids. NMR studies have shown that zinc finger motif is a simple ββα fold. The two cysteines are positioned close to β-turn and the two histidines are in the C-terminal portion of α-helix. Binding of the Zn ion cause the bending of β antiparallel sheets more close to the α-helix to form a compact structure. Each motif recognize 3-4 bp. The α-helix fits into a major groove where most of the base contacts are made. Structural studies have shown that only few key residues on α-helix are crucial for sequence recognition. Many experiments have shown that by changing amino acid in key residues the specifity of the finger is altered, which greatly simplifies further predictions for recognition of novel DNA sequences.<ref name="ref3">URNOV, F. D., MILLER, J. C., LEE, Y. L., BEAUSEJOUR, C. M., ROCK, J. M., AUGUSTUS, S., JAMIESON, A. C., PORTEUS, M. H., GREGORY, P. D. & HOLMES, M. C. 2005. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature, 435, 646-51.</ref>

Zinc finger motifs are found in tandem assembling of different motifs, which allow precise control over the sequence specificity of the protein. The main goal of today’s researches is to design zinc fingers that will bind to pre-determined DNA sequence. Scientists use different methods for determining specificity of the motif, but the far most used technique that also generated thousands of selctive zinc fingers is phage display. This technique helped to reveal some principles about zinc-finger-DNA recognition. For example, if the first base on the 5’-end of a chosen sequence is guanine then almost certain amino acid at the key residue would be arginine.<ref name_"ref4">JAMIESON, A. C., MILLER, J. C. & PABO, C. O. 2003. Drug discovery with engineered zinc-finger proteins. Nat Rev Drug Discov, 2, 361-8.</ref>

In general, zinc finger engineering methods are divided in two groups: modular assembly and alternative approaches. Modular assembly involves assembly of precharacterized single zinc finger modules as it is shown in [http://www.nature.com/nrd/journal/v2/n5/fig_tab/nrd1087_F1.html FIG 1] This method has an efficay rate less than 6 % but it is easy to preform. On the other hand alternative approaches involve combinatorial selection-based methods that result in multifinger domain with high specifity and affinity to targeted sequence. The major disadvantage of this method is the usage of large randomized libraries and not all the laboratories possess required expertise.<ref name="ref5">MAEDER, M. L., THIBODEAU-BEGANNY, S., OSIAK, A., WRIGHT, D. A., ANTHONY, R. M., EICHTINGER, M., JIANG, T., FOLEY, J. E., WINFREY, R. J., TOWNSEND, J. A., UNGER-WALLACE, E., SANDER, J. D., MULLER-LERCH, F., FU, F., PEARLBERG, J., GOBEL, C., DASSIE, J. P., PRUETT-MILLER, S. M., PORTEUS, M. H., SGROI, D. C., IAFRATE, A. J., DOBBS, D., MCCRAY, P. B., JR., CATHOMEN, T., VOYTAS, D. F. & JOUNG, J. K. 2008. Rapid "open-source" engineering of customized zinc-finger nucleases for highly efficient gene modification. Mol Cell, 31, 294-301.</ref> There are several methods under investigation that utilize different systems like, yeast two hybrid system, bacterial one- and two- hybrid system, etc.

===Cleavage domain===
Cleavage domain that is typically used is taken from the type IIs restriction endonuclease FokI. It is fused to the C-terminus of zinc finger domain with a 6 bp long linker that connects them. Cleavage of the targeted DNA sequnce is initiated only when nucleases dimerize. This method typically requires two distinct ZFN subunits to bind as a heterodimer at a desired cleveage site. However, homodimers can be also formed but they present problem because they do not cleave at a desired site. Scientists also observed that at high ZFN concentration, dimerization can procede without previous binding to the target sequence and that also contributes to the so-called off-target effect. <ref name="ref1">MILLER, J. C., HOLMES, M. C., WANG, J., GUSCHIN, D. Y., LEE, Y. L., RUPNIEWSKI, I., BEAUSEJOUR, C. M., WAITE, A. J., WANG, N. S., KIM, K. A., GREGORY, P. D., PABO, C. O. & REBAR, E. J. 2007. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol, 25, 778-85.</ref> <ref name="ref6">SZCZEPEK, M., BRONDANI, V., BUCHEL, J., SERRANO, L., SEGAL, D. J. & CATHOMEN, T. 2007. Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases. Nat Biotechnol, 25, 786-93.</ref> 

Several different protein techinques were studied in modifying nuclease domain to minimize off-target effects. For example, in one study scientists modify the dimerization interface so that only expected heterodimers were active.<ref name="ref6">SZCZEPEK, M., BRONDANI, V., BUCHEL, J., SERRANO, L., SEGAL, D. J. & CATHOMEN, T. 2007. Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases. Nat Biotechnol, 25, 786-93.</ref>

Even though ZFNs as reagents used in gene therapies are not optimal yet there are lots of experiments that use them in treating certain diseases. Recently, an experiment was published that use ZFNs to modify genome, which resulted in HIV-1 resistance strain. 

==HIV==
The human immunodeficiency virus (HIV) is a lentivirus that causes the acquried immunodeficiency syndrome (AIDS). Viruses transduce their viral core with (+) single-stranded RNA into the host immune cells such as helper T cells (CD4+ T cells), macrophages and dendritic cells. This results in decreased number of CD4+ T cells and subsequent diminished immunity of the infected person. Patients are more susceptible for further infections, which are usually lethal. 

There are two types of HIV: HIV-1 and HIV-2. HIV-1 is responsible for majority of HIV infected individuals worldwide. It is more virulent and infective than HIV-2, which is less easily transmitted and the time between initial infection and illness is longer. HIV-1 can be further classified upon its phenotype. The name of the isolate is based on the type of co-receptor that together with CD4 govern entry of HIV-1 to target cells. Numerous studies have shown that CCR5 and CXCR4 are the major co-receptors found in HIV-1 strains. Isolates that use only CCR5 are termed R5 viruses and if they use only CXCR4 are termed X4 viruses.<ref name="ref7">BERGER, E. A., DOMS, R. W., FENYO, E. M., KORBER, B. T., LITTMAN, D. R., MOORE, J. P., SATTENTAU, Q. J., SCHUITEMAKER, H., SODROSKI, J. & WEISS, R. A. 1998. A new classification for HIV-1. Nature, 391, 240.</ref>

===HIV-1 co-receptors===
Both co-receptors (CCR5 and CXCR4) belong to a large protein family of G protein-coupled receptors that participate in signal transduction. However, HIV also uses them to enter the host cell through the interaction between viral envelope glykoproteins and plasma membrane receptors. Gp120 and gp41 are glycoproteins that are exposed on the surface of the viral particle. Three gp120s and gp41s are combined to form a trimer of heterodimers, thereby creating an envelope spike for viral entry. Gp120 binds CD4 receptor on CD4+ cells and a large binding energy that is relesed drives conformational changes that expose a co-receptor binding site on gp41. Eventually, conformational changes within gp120/gp41 lead to the fusion of membranes.<ref name="ref8">DRAGIC, T. 2001. An overview of the determinants of CCR5 and CXCR4 co-receptor function. J Gen Virol, 82, 1807-14.</ref>
 
Studies have shown that R5 isolates are sexually transmitted and persist within majority of infected individuals. However, after several years of infection the phenotype is converted from R5 to X4, which is also a sign of the disease progression.

Scientists observed that some individuals who were frequently exposed to HIV remained uninfected. Further investigations have shown that homozygous Δ32 mutation in CCR5 confers resistence to HIV-1. Deletion of 32 bp causes a frameshift that truncates CCR5 and prevents its expression on the plasma membrane. Heterzygous for the CCR5/Δ32 allele are not resistent to HIV but it was shown that it have a protective effect on early disease progression. Since that discovery, a development of drugs that target virus-CCR5 interaction has emerged. There are four groups of agents that target CCR5 co-receptor function.  Monoclonal antibodies bind to gp120, thereby inhibit binding to CCR5 or they prevent virus fusion and entry. Chemokines inhibit fusion and entry by blocking gp120 binding to CCR5. Peptides and small molecules inhibit HIV-1 replication by disruption of helix-helix interaction in CCR5<ref name="ref8">DRAGIC, T. 2001. An overview of the determinants of CCR5 and CXCR4 co-receptor function. J Gen Virol, 82, 1807-14.</ref>. However, therapies that use small molecules have resulted in development of resistance. Recent strategies that are being used are based on long-term genome editing. One of the experiments that use this approach is based on ZNF and will be described below.

==Establishment of HIV-1 resistance in CD4+ T cells by ZFNs==
Group led by Carl H. June designed ZFNs to distrupt endogenous CCR5 of primary human CD4+ T cells, thereby generating a HIV-resistant genotype ''de novo''. Their goal was to engineere ZFNs, which will bind to specific sequence in the CCR5 coding region upstream of the natural CCR5/Δ32 mutation resulting in permanent disruption of CCR5 in CD4+ T cells. The experiment was carried out ''in vitro'' as well as ''in vivo'' in a NOG model mouse of HIV infection. They have shown that genome modification by CCR5 ZFNs confers robust protection against HIV-1 infection.<ref name=<ref9">Cys2His2 zinc finger proteins. Annu Rev Biochem, 70, 313-40.
PEREZ, E. E., WANG, J., MILLER, J. C., JOUVENOT, Y., KIM, K. A., LIU, O., WANG, N., LEE, G., BARTSEVICH, V. V., LEE, Y. L., GUSCHIN, D. Y., RUPNIEWSKI, I., WAITE, A. J., CARPENITO, C., CARROLL, R. G., ORANGE, J. S., URNOV, F. D., REBAR, E. J., ANDO, D., GREGORY, P. D., RILEY, J. L., HOLMES, M. C. & JUNE, C. H. 2008. Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. Nat Biotechnol, 26, 808-16.</ref>

===Results===

====Design of CCR5 ZFNs====
They engineered and optimized a large series of ZFNs that target CCR5. After optimization they chose zinc finger proteins that contains four zinc finger motifs, thereby recognizing 24 bp in all. The target sequence that is shown in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F1/ FIG 1a] was located upstream of the normal Δ32 mutation assuming that this location would display substantial structural sensitivity of the CCR5 protein. They reasoned that repair of DSB via NHEJ following nucleases activity would result in truncated or nonfunctional gene products and would not be expressed on the cell surface. The zinc finger domain was coupled to the DNA cleveage domain of FokI and it is referred as ZFN-215. They also designed another construct that contains modified cleveage domain to function as obligate heterodimer referred as ZFN-224.

====Entry inhibition of R5 isolates by ZFNs targeted-disruption====
The research group determined wheather trunsduction with an adenovirus (Ad5/35) vector of GHOST-CCR5 cells with CCR5 ZFNs would alter CCR5 expression on the cell surface and HIV-1 entry. GHOST-CCR5 cells were used as they represent reporter cell line for HIV-1 infection. This cell line contains numerous copies of CCR5 expression casettes and an inducible green flourescent protein (GFP) marker gene under the control of the HIV-2 long terminal repeat. Viral entry assumedly depends on the presence of CCR5 receptors on the cell surface. If the CCR5 is present on the membrane the virus can enter the cell, thereby ativates GFP expression. The group confirmed and quantified the generation of ZFN-induced mutations on the target site. They used an assay based upon the mismatch-sensitive Surveyor nuclease. The assay showed high efficacy in mutation on targeted sites (50-80 %) as it is shown in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F1/ FIG 1b]. They used two controls: nontransduced control cells and cells transduced with the same vector although it encodes interleukin (IL)-2Rγ-specific ZFNs instead of CCR5 ZFNs.

After one week the cells were infected with HIV-1BAL, which is a prototype of R5 isolates. They analyzed CCR5 surface expression using flow cytometry and found to be reduced by more than tenfold in the cells that were transduced by CCR5 ZFN compared to the control (IL)-2Rγ-treated cells as is shown in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F1/ FIG 1c]. Additionally, these results were consistent with the expression of GFP, which was reduced in cells that were treated with CCR5 ZFNs [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F1/ FIG 1d]. These results demonstrate that CCR5 ZFNs can efficiently cleave their DNA target site in CCR5, thereby preventing CCR5 expression on the cell surface, which results in resistance to R5 isolates infection.

====Survival advantage of ZFN-modified CD4+ T cells in vitro====
Genome editing that results in genetic change of CCR5 allele should confer a long-term resistance to HIV-1 so this was their next experiment. PM1 cells that resambles to CD4+ T cells in the CCR5 expression were electroporated with CCR5 ZFNs expression plasmids. DNA tests from the population of trated cells were preformed and the results showed a distruption level 2,4 % of an endogenous CCR5. After a week cells were infected with HIV-1BAL or mocked infected and they were expanded in continous culture for 70 days. DNA test was preformed at different time points and it had shown that by day 52 of infection the HIV-1BAL infected PM1 cells had undergone 30-fold enrichment for ZFN-modified CCR5 alleles compared to cells that were mocked infected as it can be seen in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F2/ FIG 2a]. These results indicate that HIV-1 provides a potent selective pressure for CCR5 ZFN-modified cells.

They also determine the CCR5 ZFN-mediated mutations by sequencing the CCR5 ZFN target region. They found numerous distinct short deletions and insertions in 78 % of sequence reads, shown in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F2/ FIG 2b]. However, more than 30 % of all modified sequences contained specific 5-bp insertion, which resulted in introduction of two stop codons, generating a trunction of the wild type protein.

====Selective advantage of ZFN-modified primary CD4+ T cells during HIV-1 infection====
The group also detrmined the efficacy of CCR5 ZFNs-mediated modifications in primary human CD4+ T cells. Cells were collected from healthy wild-type CCR5 donors and were transduced with Ad5/35 vector encoding CCR5 ZFNs. The results from Surveyor assay had shown that 40-60 % of the CCR5 alleles undergone disruption, as it is seen in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F3/ FIG 3a]. There was no difference in population-doubling rate between the modified CD4+ T cells and nontransduced cells [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F3/ FIG 3b].

Scientists had shown that CCR5 ZFNs-transduced CD4+ T cells infected with R5 isolates resulted in stability and twofold enrichment of gene-edited cells [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F3/ FIG 3c]. On the other hand, the same cells transduced with Ad5/35 GFP control vector did not show detectable disruption.

They also determined the percentage of CD4+ T cells that were modified in both alleles. They found out that 33 % of the cells that had CCR5 modification (23 % of the whole population) were homozygous for CCR5 disruption. They reasoned that with selective presure the frequency of homozygous disruption would be higher.

====Specificity of CCR5 ZFNs in primary CD4+ T cells====
Genome editing with ZFNs is the potential clinical application for curing certain diseases, so it is critical to verify the efficacy, tolerability and specificity of ZFN action. Scientists did this with three different approaches: the 53BP1 immunostaining, the Surveyor nucleases and the 454 pyrosequencing data. 

53BP1 is protein that is recruited to the sites of DBSs and is required for NHEJ. CD4+ T cells were transduced with Ad5/35 vector encoding ZFN-224. Furthermore, the genomic integrity was assesssed at different time points by immunodetection of the 53BP1. This assay is based on enumeration of the number of 53BP1 foci per nucleus. They had shown 1.4-1.6-fold increase of intranuclear 53BP1 foci when compared to the controls, which are nontransduced or GFP-transduced CD4+ T cells [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F3/ FIG 3d].

To verify the specificity of ZFN-224 action, scientists experimenatlly determined the consensus ZFN binding sites by Systematic Evolution of Ligands by EXponential enrichment (SELEX). They identified 15 putative alternate cleveage sites. Moreover, Surveyor nucleases assay had shown only one site besides intended target seequence that is recognized buy ZFN-224. This site is positioned in CCR2 alleles. CCR2 is homolog of CCR5 and it cannot be immunodetected with 53BP1 because they are in close proximity. However, a small proportion of modified CCR2 in CD4+ T cells is unlikely to be destructive since it had been correlated with delayed progression to AIDS in HIV-infected individuals.

Another method, the 454 pyrosequencing, was used to determine the ZFN-224 specificity. It is more sensitive than the other two in detecting rare off-target effect. Scientists designed PCR probes for all 15 sites identified by SELEX. Under conditions where CCR5 was modified at 36 % efficiency, they observed 5,39 % off-target sites on CCR2 locus and rare targeted sites (1/20000) on ABLIM2 alleles. Taken together these results, ZFN-224 are specific in CD4+ T cells.

====Reduced viremia and selection of CCR5 ZFN-modified primary CD4+ T cells during HIV-1 infection in vivo====

NOG mouse model is a generation of highly immunodeficient mouse and it is often used for researches of cancer and AIDS. In this study, scientists used this model to explore feasibilty, safety and therapeutic potential. Primery CD4+ T cells were transduced with Ad5/35 vectors encoding the CCR5 ZFNs or GFP (control). Those cells were expanded and then transplanted to the NOG mice either noninfected or HIV-1–infected phytohemagglutinin A (PHA)-blasted periheral blood mononuclear cells (PBMC) [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F4/ FIG 4a]. Peripheral blood was taken on the indicated days after adoptive transfer and analyzed for human CD4, CD8 and CD45 expression. They observed reduced CD4+ to CD8+ cell ratio in HIV-infected groups.

Mice were killed after a month from HIV infection and the genomic DNA from human CD4+ T cells was purified for further analysis. They evaluated the efficacy of CCR5 ZFNs-mediated disruption with Surveyor nucleases assay as it can be seen in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F4/ FIG 4b]. They found 27,5 % average CCR5 disruption in ZFNs-treated CD4+ T cells, compared with animals that got the same amount of starting population CD4+ T cells in the absence of HIV infection [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F4/ FIG 4c]. Another independent experiment had shown the protective effect that CCR5-modified cells had on CD4+ T cells depletion and on viremia. The experiment was similar to the one described above but it was followed for 50 days. The number of CD4+ T cells had increased in days from 30-50 and 8 of 10 HIV-infected mice had more than 50 % CCR5-modified CD4+ T cells [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422503/figure/F4/ FIG 4d]. Taken these results together, the modified cells confer resistance to HIV-1 infection ''in vivo''.

==Conclusion==
In conclusion, presented results support clinical development of adoptive immunotherapy in treating HIV-1 infected individuals by reconstituting CD4+ T cell pool to CCR5 null genotype with ZFNs. It was shown that ZFN-mediated genome modification of CD4+ T cells was highly specific, well tolerated and stable as was revealed by various experiments that were carried out ''in vitro'' as well as ''in vivo''. However, despite good results that were presented here there are some other challenges to be concered when applying ZFN-mediated genome editing to clinical therapies. Firstly, as it was shown ZFN-induced repair process result in diverse range of deletions and insertions. Some of them could be result of a novel CCR5 epitopes and further elimination by the host immune system. Secondly, animal models that ware used in this research are valid only for studying resistance to infection and do not present conditions that usually persist in HIV-infected individuals. Nonetheless, genome editing with ZFNs eliminates viral entry without the integration of any foreign DNA into the genome. Finally, recent work pinpoints that it is possible to use ZFNs-based approaches in stem cells, thereby it cloud be applied to a number of monogenic diseases.


==References==
<references/>