neighboring loci. Therefore, the interaction between enhancers and promoters in the transgene and the locus and neighboring areas should be as minimal as possible. Safe harbor sites could be allocated in genes or in genetic deserts. Both strategies present advantages and disadvantages. The main advantage of considering a locus as a safe harbor is that the chromatin conformation in this area is usually opened, allowing the expression of the integrated transgene. However, the transgene incorporation could trans-activate the endogenous genes or deregulate its expression due to the disruption of a regulator area in the intron in which they are usually incorporated. Moreover, genes that could be used as safe harbor locus should be expressed widely all over cell types or at least in the cells of interest. On the other hand, genetic deserts do not present in principle, the problem of transactivation or deregulation of endogenous genes, although these areas could contain regulatory regions driving expression of very distal genes. The main limitation of these areas is that the chromatin is usually very compacted, making gene targeting more difficult and also reducing the expression of the integrated transgenes. Moreover, potential conformational changes produced in the chromatin in these areas after gene editing should be taken into account in terms of a possible deregulation of the cell status.
Safe harbor sites should be functionally validated, enhancing the effect of the insertion in neighboring genes in vitro, and determining possible changes in the epigenetic marks in the locus after the introduction of the transgene. Finally, these in vitro studies should be confirmed in vivo to conclude that the targeting in this locus does not produce any alteration in the tissue and in the organism, including risks of malignant transformation.
The two safe harbor most frequently used are the chemokine (C-C motif) receptor 5 (CCR5) and the adeno-associated virus site 1 (AAVS1):
a) CCR5: is located in chromosome 3 (3p21.31) and codifies the principal co-receptor of the HIV-1. The first evidence that pointed out that this locus could act as a safe harbor came from the resistance of individuals to HIV-1 infection, due to a null mutation in this gene (CCR5Δ32)61. The mutation of this gene did not induce any pathology and has
been already used in the first clinical trial using nucleases to treat HIV-1 patients; in which no side effects have been observed so far62. The endogenous expression of this gene is variable along cell types: high in T lymphocytes, monocytes and macrophages, but it is not expressed in B lymphocytes or dendritic cells63. Moreover, the knockout
mice for Ccr5 presents impaired leukocyte migration and increased susceptibility to some infections64. Another disadvantage of this locus is that the expression of a
reporter gene, GFP, in T cells is lower in this site as compared with the expression of the same reporter gene in another safe harbor locus: the AAVS165. Furthermore, the high homology of this locus with the CCR2 gene make the design of nucleases more complicated increasing the off-target events66.
b) AAVS1: is located in chromosome 19 (19q13.42). It was described as the common integration site of the adeno-associated human virus67, which disrupts the protein
phosphatase I regulatory inhibitor subunit 12C (PPP1R12C). This gene codifies a protein whose function is not well understood, and no side effects have been
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described derived from its interruption. Moreover, it has been described that approximately 80% of the population presents detectable levels of antibodies against some AAV serotypes, which means that they probably harbor integrations of this virus in the AAVS1 locus without any pathogenic or side effect68. This region has been used in gene targeting experiments in T-cells, neural stem cells, embryonic stem cells, fibroblasts and iPSC69-75. These studies support a stable and robust expression of the different transgenes integrated in the AAVS1 locus all over the different cell types. Moreover, it has been demonstrated that the integration of a transgene in this locus does not induce any adverse effect65. The chromatin of this locus has an open and
active conformation, and the insertion in the AAVS1 locus does not alter the expression of surrounding genes, probably due to the presence of an insulator, which prevents the transactivated expression of surrounding genes76. All these characteristics make the AAVS1 locus as the best safe harbor locus for gene targeting approaches described up to now.
2.3. Designed nucleases
Nucleases are the tools that generate the DSBs in a specific locus in the genome for gene targeting. There are four different types of nucleases that have been widely used in gene targeting: meganucleases (MGNs), zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered, regularly interspaced, short palindromic repeat associated to Cas9 nuclease (CRISPR/Cas9) system (Figure 7). All of them possess a DNA recognition region and a catalytic domain which generates the DSB in a specific locus.
Figure 7: Different types of nucleases. MGN: meganuclease. They recognize long sequences of DNA cutting by homology. ZFN: zinc-finger nuclease. They are formed by two subunits: homology domain composed by groups of three ZF domains which recognize a specific nucleotide and the catalytic domain
MGN
ZFN
TALEN
CRISPR/Cas9 gRNA
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