University of Groningen

Epigenetic reprogramming of endogenous genes for permanent modulation of geneexpressionHuisman, Christian

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General discussion and future perspectives

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General conclusion and discussion

The ultimate aim of this thesis was to regain control over the transcriptome in disease by sustainable epigenetic reprogramming. First, we validated the dysregulaton of target genes involved in the pathology of cancer in (primary) cell lines or patient material. Subsequently, we engineered artificial transcription factors (ATFs) to modulate the endogenous expression levels of the selected genes in disease models and studied the functional effects. Finally, we aimed to overwrite the epigenetic signature (epigenetic editing) of the target genes by zinc finger proteins (ZFPs) (from the validated ATFs) connected to epigenetic enzymes to obtain long term transcriptional modulation. The role of EpCAM in ovarian cancer The first gene we set out to target with ATF technology was EpCAM (chapter 2). EpCAM is overexpressed on various cancer types, such as breast, ovarian and renal cancer (1). The biological role of EpCAM overexpression in cancer is paradoxical; EpCAM overexpression can be associated with improved or worse patient survival (1). For ovarian cancer, the role of EpCAM overexpression is unclear and was addressed in this study by bidirectional EpCAM modulation using ATFs. The ATFs were obtained from a previous study which showed EpCAM expression modulation from a co-transfected luciferase construct carrying the EpCAM promoter (2). In the current study, we could also modulate endogenous EpCAM levels, most likely because of more efficient delivery of the ATF. In a panel of breast and ovarian cancer cell lines EpCAM RNA and protein levels were significantly modulated by ATFs. ATF-mediated downregulation of EpCAM in breast cancer cells confirmed the oncogenic function of EpCAM in this malignancy, as downregulation resulted in growth inhibition. Previouly, siRNA-mediated downregulation of EpCAM has shown to reduce the metastatic potential of breast cancer cells (3). In ovarian cancer, however, ATF-induced repression did not result in less viability of the cells or a decrease in growth rate. Also siRNA-mediated downregulation of EpCAM (used as control) did not affect the growth of the cells. On the other hand, ATF-mediated upregulation/re-activation or overexpression of EpCAM cDNA decreased cell growth, indicating a tumor suppressive role for EpCAM in this ovarian cancer cell line. Note that the induced tumor suppressive effects by EpCAM overexpression could neither be rescued by repressive ATFs nor siRNA mediated downregulation. A possible reason for this observation is that the initial EpCAM hit by the ATFs harmed the cells in such a way that recovery could not be achieved. Taken together, this study demonstrated that EpCAM inhibition does not offer a promising therapeutic approach for ovarian cancer.

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ICAM-1 as tumor suppressor in ovarian cancer As the potentials of ATFs in cancer with respect to re-activation of tumor suppressor genes (TSGs) have been demonstrated by the induction of Maspin (4) and EpCAM, we further explored the potentials of ATF-technology for the re-activation of silenced ICAM-1 (chapter 3). Although ICAM-1 is not known for its tumor suppressive effect, its expression is significantly downregulated in ovarian cancer (5). Whereas ICAM-1 expression on tumor cells is of importance for attracting immune cells, ICAM-1 might also directly induce cell growth inhibition upon re-expression. Indeed, upregulation of ICAM-1 by ATFs resulted in reduced cell growth of ovarian cancer cells, also in the absence of immune cells. Moreover, ICAM-1 negative ovarian cancer cell line showed an extensive apoptotic response associated with ATF-induced ICAM-1 expression. These results are in line with previous reports which demonstrated that ectopic expression of ICAM-1 cDNA decrease tumorigenicity in vivo (6, 7). However, the reduced growth effects of ICAM in vivo have so far been assigned to the evoked immune response by ICAM-1 overexpression (8). Here, we demonstrated a direct cellular role of ICAM-1 as TSG without the presence of immune cells. Taken together, this study showed that, next to its established immunogenic role, ICAM-1 affects cell biological behavior of ovarian cancer cell lines. We demonstrated that ATF-technology presents a powerful approach for elucidating gene-function in a pathologic environment.

Diagnostic marker genes in cervical cancer In the next three chapters, we explored the potentials of biomarker genes in cervical cancer as therapeutic targets for this malignancy. As DNA hypermethylation is often observed for silenced TSGs, hypermethylated genes currently extensively explored as biomarkers, might exhibit tumor suppressive activities upon re-expression. Previously, our group identified C13ORF18, CCNA1, TFPI-2 and EPB41L3 as gene promoters specifically methylated in this cancer and not in normal cervical cells (9, 10). In chapter 4, we aimed to re-express C13ORF18, and studied the putative tumor suppressive actions of this gene after ATF-mediated re-expression. The engineered ATFs were able to re-activate C13ORF18 with induction levels up to 130-fold in methylated cells, which resulted in a less malignant phenotype of the cells. Interestingly, the strongest re-expressor, was able to induce C13ORF18 levels equal to or higher than endogenous levels observed for unmethylated cells. This re-expression of C13ORF18 in HeLa was associated with a strong apoptotic response and a strong decrease in cell proliferation. Contrary, human fibroblast cells tranduced with the same constructs did not show any signs of distress, which may indicate a cancer specific role for C13ORF18. The data in this study convinced us to assign a tumor suppressive role to C13ORF18 in cervical cancer.

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We also observed synergy of C13ORF18-targeting ATFs with epigenetic drugs (5-aza-dC or TSA). Previously, this was also observed for Maspin-targeting ATFs, where epigenetic drugs enhanced Maspin re-expression several folds (11). Such synergistic effects of ATFs with epigenetic drugs were also observed for targeting the epigenetically silenced Oct4: only co-treatment with epigenetic drugs allowed activation of this gene to significant levels (12). Interestingly, such synergistic effects were not observed for ATFs targeted to frataxin (13). Here, we also showed that non-functional C13ORF18-ATFs, did show an effect when co-treated with epigenetic drugs. Possibly, epigenetic drugs relax the hypermethylated C13ORF18 promoter, enabling also ineffective ATFs to bind to their aimed target sites. Indeed, we showed increased binding of the ATFs to the C13ORF18 promoter after treatment with epigenetic drugs. Another reason for the synergistic effects of ATFs with epigenetic drugs may be that 5-aza-dC or TSA increases the gene-activating potential of VP64. Furthermore, in this study we found that ATF-mediated re-activation of C13ORF18 affected the local epigenetic signature (DNA methylation and histone modifications). First evidence about the potency of ATFs to transiently affect epigenetic components appeared in a study on Maspin-ATFs in 2011 (14). This study demonstrated DNA demethylation by two Maspin-ATFs (carrying a VP64-effector domain) in the Maspin promoter. Interestingly, this was the first study that accomplished targeted DNA demethylation. The orientation of the effector domain was of great influence for the observed effect; DNA demethylation occurred in the direction of the effector domain and was absent on the side of the ZFP. The site-specific effects indicate a role for VP64 in the DNA demethylating process, although the exact mechanism is unknown. Interestingly, we also found that treatment with our ATFs resulted in some demethylation of the targeted promoter. Recently, this demethylating potential of VP64 was further confirmed by the targeting of ICAM-1 in ovarian cancer cells (15). Especially around the ZFP-target site, a decrease in DNA methylation level could be detected. Furthermore, they found that the ZFP without VP64 can induce DNA demethylation at the target site as well (16). This demethylating effect of ZFPs was also reported for the Maspin-ATFs at the target site (14). A possible reason for this may be competition with Dnmt1, due to steric hindrance by ZF binding. As DNA methylation and histone methylation are closely linked to each other (17), we also investigated any effect of the ATFs on the histone code. Indeed, we found that the loss of DNA methylation (or increase of gene expression) was accompanied with a decrease in the repressive histone mark H3K9Me3. This epigenetic modification fits the description of a more active promoter, and proves that ATFs are capable of temporally changing the histone code, at least to some extent. Around the same time, also another group reported on the ability of ATFs

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to affect histone marks; however this study was done in stable transfectants cultured for several weeks (18). Epigenetic editing of genes in cervical cancer The successful targeting of endogenous genes using VP64- or SKD-ATFs opens the way to target a variety of epigenetic enzymes for epigenetic rewriting of promoter regions (Epigenetic Editing, Figure 1). Of most importance would be the targeting of DNA (de)methylases; VP64 or SKD-ATFs act transiently on gene expression and genes tend to return to the ‘normal’ state after the ATF is removed (19, 20). Although ATFs induce a transient change of the epigenetic state as shown in chapter 4, these changes are likely insufficient to have a sustained effect. Direct epigenetic reprogramming by DNA (de)methylases is a way to improve sustainability of induced effects, and to prolong induced alterations of cell fate in the future. In chapter 5, we described an efficient approach to re-activate hypermethylated candidate tumor suppressor genes by ten-eleven translocation methylcytosine dioxygenase 2 (Tet2)-induced demethylation in cervical cancer cell lines. First, we studied the mRNA expression levels and epigenetic regulation of four genes in a panel of cervical cancer cell lines and found that CCNA1 and C13ORF18 were strongly silenced in most cell lines by DNA methylation and repressive histone marks, and to a lesser extend TFPI2. This observation is in accordance with the observed methylation of these genes in cervical cancer patient samples compared to healthy controls. Next, we showed that these hypermethylated genes (C13ORF18 and CCNA1) can be efficiently demethylated by Tet2, resulting in gene re-activation. Demethylation of single CpGs was observed with reduction up to 38% for C13ORF18 and 59% for CCNA1, resulting in gene-inductions (up to ~57- and ~80-fold respectively). Tet2-induced demethylation was very pronounced at both sides of the binding site (within 50 bp), however the bisulfite sequencing data for C13ORF18 suggest a much further spreaded effect at the side of the demethylase. Expression of VP64-ZFPs had only limited effect on DNA methylation. Therefore, the described strategy to remove methylation by Tet2 seems a promising approach to get rid of unwanted DNA methylation at TSG promoters. Previously also targeting of Tet1 to the HOXF2 and beta-globin genes resulted in efficient demethylation and upregulation of targeted genes (21), while targeting of Tet2 to the ICAM1 promoter resulted in a twofold gene induction (16). Using various growth assays in among others the tumor-derived cell line CC-11, we identified TFPI-2 as the strongest growth reducer from the explored genes in this chapter (C13ORF18, CCNA1, TFPI2 and Maspin). TFPI-2 is identified as TSG in several types of cancer (22-24) and here we also observed that TFPI-2 possesses tumor suppressive activities in cervical cancer cell lines; both after ATF-mediated overexpression, as well as after cDNA overexpression. Therefore, TFPI-2 may represents a novel therapeutic target in cervical cancer. Maspin was the least

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effective therapeutic target for this malignancy as Maspin re/over-expression did not decrease cell growth. Strikingly, Maspin was reported as silenced gene in cervical cancer (25, 26), but we found high expression of Maspin in cervical cancer cell lines as well as in tumor specimens. Therefore, we concluded that Maspin does not represent a therapeutic target in cervical cancer for gene-induction. Furthermore, the data in this study demonstrated that ATF-mediated up- or downregulation achieved similar effects on gene expression and functionality as a traditional technique like cDNA overexpression. Silenced gene by DNA methylation and repressive histone marks Forced re-expression by zinc finger proteins linked to effector domains

Figure 1. Epigenetic Editing. Epigenetic editing refers to the targeted overwriting of epigenetic marks to permanently reprogram gene expression profiles. Self-engineered gene-targeting ZFPs are coupled to effector domains, which overwrite the epigenetic marks in the targeted promoter. Also in chapter 2 we found that EpCAM cDNA overexpression by transient transfection induced similar effects as ATF-mediated re-expression. One of the disadvantages of cDNA overexpression is that only one splice variant is expressed. In contrast, ATFs have shown to allow expression of multiple splice variants as expression is mediated from the endogenous gene locus (27). As gene inductions

methylation CpG unmethylation CpG histone acetylation histone methylation

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by the ATFs found in this study are substantially less compared to cDNA overexpression, the similar phenotypic outcome may have been the result of the more efficient (all splice variants) gene inductions obtained by the ATFs, although this hyposthesis was not tested in this study. A promising strategy to further increase gene re-expression is by simultaneously targeting multiple regions in one promoter by different ZFP-VP64 constructs. In one study, four ZFPs fused to demethylator thymine DNA glycosylase (TDG) removed methylation more efficiently than a single targeted construct (28). The synergistic effects between constructs were also observed by simultaneously expressing of multiple CRISPR/Cas9-VP160 (29), TALE-VP64 (30), TALE-Tet1 (21) or ZFP-p65 (31) fusion constructs/proteins targeted to a single promoter, resulting in improvements compared to expression of a single fusion. Also the activation of VEGF-A by different activation domains showed such cooperativity, which was also observed for the downregulation VEGF-A using different repressive domains. In addition, chapter 5 showed that ZFP-VP64 ATFs revealed such synergistic effects as well, as 3ab-VP64 and 5ab-VP64 more strongly increased re-expression of C13ORF18 than single treatment. Kinetics of ATF-mediated re-expression can be sustained with epigenetic drugs In chapter 6 we aimed to fully reprogram silenced promoters in order to improve the sustainability of gene re-expression by targeting EPB41L3 in the presence of epigenetic drugs in cervical cancer cell lines. EPB41L3 has been reported as TSG in a range of different malignancies, including breast- and ovarian cancer and therefore qualifies as an important therapeutic target (32-34). Based on its silenced state in cervical cancer, we suspected that EPB41L3 may represent a therapeutic target in cervical cancer as well. Interestingly, in a recent study EPB41L3 was appointed as the most potent biomarker for the early detection of cervical cancer (35, 36). First, we demonstrated the association between promoter hypermethylation and EPB41L3 expression, and found that the expressing cell lines showed lower degrees of promoter methylation. However, this relation turned out to be less straightforward as for the C13ORF18 gene. Strikingly, the highest expressing EPB41L3 cell line (SiHa) turned out to be completely hypermethylated across two CpG islands in its promoter. This observation is inconsistent with the bulk of literature, as methylated genes are typically silenced (17, 37, 38). hTERT is one other example of a gene in which increased methylation in many cancer types results in upregulation of expression (39), most likely because of the preservation of unmethylated DNA and active chromatin around the transcription start site (TSS) (40). For EPB41L3, the CpG island is located downstream of the TSS; therefore, DNA hypermethylation of EPB41L3 promoter may less a determing factor for EPB41L3 expression compared to genes with the TSS situated in the CpG island. Factors associated with active gene transcription are not hampered by repressive complexes found on

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hypermethylated DNA, and therefore gene transcription may be preserved although the CpG island is hypermethylated. Also other studies have shown that single CpG methylation can have major influence on gene transcription, for example CpGs in the HBB promoter (21). Two ATFs targeting the EPB41L3 promoter in close proximity to the TSS were able to upregulate EPB41L3 in seven tested cancer cell lines (breast, ovarian and cervix), even in highly methylated (silenced) cell lines. Therefore, this study demonstrated the potentials of the EPB41L3-ATFs for re-expression of this gene in a variety of epigenetic landscapes. Using ATF-technology, we demonstrated the tumor suppressive activities of EPB41L3 in ovarian- and breast cancer cell lines and validated the hypothesized tumor suppressive role of EPB41L3 in cervical cancer. Untill now, little is known about the sustainability of gene modulation by VP64-ATFs. As ATFs do change epigenetic marks (DNA methylation and histone marks), re-expression may be sustained. One earlier study, however, showed that re-expression of target gene Maspin was found to be sustained only for a few days, as Maspin expression levels declined shortly after removal of the Maspin-ATF (19). Such short-term effects were also observed for downregulation of Maspin by SKD-ATFs. However, long-term stable repression could be observed when the Mapsin-ZFP was fused to DNA methyltransferase 3a (41). This was the first study demonstrating that site-specific DNA methylation by targeting epigenetic enzymes to an endogenous gene is feasible. Such approach reprograms promoter regions which can induce long-term effects on gene-expression. For gene re-expression, co-treatment of VP64-ATFs with epigenetic drugs may increase the sustainability of re-expression. The synergistic actions of epigenetic drugs and ATFs may allow more complete reprogramming of silenced promoters compared to ATFs or epigenetic drugs alone, as gene re-expression by ATFs is strongly increased in the presence of epigenetic drugs. Previously, also the synergistic effects between 5-aza-dC and HDAC inhibitors have shown to increase gene-expression, and this increase in gene re-expression was associated with promoter reprogramming (42). Furthermore, the HDAC inhibitor TSA has shown to increase the sustainability of 5-aza-dC induced re-expression (43). In chapter 4, we demonstrated the synergy between ATFs and epigenetic drugs with respect to gene expression. In chapter 6, we studied whether co-treatment of ATFs with epigenetic drugs may increase the sustainability of ATF-induced re-expression. For studying the kinetics of ATF-mediated re-expression of EPB41L3 in the presence of epigenetic drugs (5-aza-dC, TSA), we made DOX-inducible cell lines. Upon DOX-treatment, we showed induction of EPB41L3, which was increased several folds by co-treatment with epigenetic drugs. While EPB41L3 expression declined after removal of the ATF, we indeed achieved sustained re-expression of EPB41L3 when cells were co-treated with both 5-aza-dC as well as with TSA for the measured five days. The synergistic effects of ATFs and epigenetic drugs on

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the chromatin landscape apparently lead to more stable reprogramming of the targeted gene promoter than ATFs or epigenetic drugs alone, allowing for the sustained gene-expression. Furthermore, our data showed that TSA facilitates re-expression of target genes by demethylating enzymes (around twofold). The exact mechanism behind the re-inforcing effects between epigenetic drugs and ATFs/epigenetic editors is largely unknown, but may partly be explained by increased binding of the ATF to its endogenous target site (chapter 4), acetylation of lysine residues within transcription factor (44)) and enhanced expression of the transgenes (45). In addition, in chapter 6, we found that TSA treatment resulted in downregulation of DNA (cytosine-5)-methyltransferase 3B (DNMT3b). TSA-induced repression of DNA methyltransferases may slow down remethylation events. DNA methylation has a rapid turnover in human cells (46), and inhibition of the process of remethylation may therefore increase the reprogramming potential of ATFs. Interestingly, we observed that expression of Tet2-fusion proteins was associated with increased expression of DNA methyltransferase DNMT3b. A possible explanation for this may be a feedback mechanism in which cells try to neutralize/antagonize decreased methylation levels induced by the demethylating reagents. It is likely that our constructs induce large scale demethylation, as ZFPs are bound to many regions of the genome (as observed by our ChIP-Seq), and thus, when fused to DNA demethylases, may cause genome-wide demethylation (47). Also TALE-targeted Tet-enzymes have shown to decrease DNA methylation levels at off-target regions (21). Similarly, large scale global DNA demethylation is observed after overexpression of untargeted Tet-enzymes (48). Such Tet-induced DNA demethylation may be normalized by the cells through increased expression of DNA methyltransferases. Epigenetic editing of the HER2 promoter It has been shown that the editing of a single mark induces spreading of the mark, thereby changing the epigenetic state of the whole region, which may result in repression of gene expression (49). In that respect, it should be possible to modulate the expression of a gene by targeting a single enzyme, which in turn, recruits other enzymes for the reorganization of the chromatin, resulting in gene expression modulation. Therefore, we explored the targeting of histone modificating enzymes (histone modifiers) to change gene expression (chapter 7). For targeting repressive histone modifiers, only few studies so far accomplished downregulation of target genes; including the downregulation of VEGF by the catalytic domains of either G9A or Suvdel76 (50), the downregulation of a number of target genes by LSD1 histone demethylase (51) and the downregulation of various neuronal genes by a variety of repressive histone modifiers (52). Here, we are the first to confirm that gene downregulation by targeting of G9A or Suvdel76 can result in substantial downregulation. The gene we targeted with repressive epigenetic editing domains was HER2 (chapter 7).

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HER2 is overexpressed in several cancer types, and targeting of this protein is widely used as anti-cancer therapy (53). In this chapter, we demonstrated that HER2-ZFPs linked to the catalytic domain of G9A efficiently induced methylation of H3K9 at the HER2 promoter. And indeed, the increase of the repressive histone mark was associated with a strong decrease in HER2 expression, almost comparable with the effects of the transient effector domain SKD. The causality between histone marks and the related gene expression is not always generally accepted. However, here we reported a clear causality of the induced histone mark on gene repression, as the targeted mutant counterpart was inactive. The observed downregulation of oncogene HER2 strongly influenced cancer cell growth as a significant decrease in metabolic activity and clonogenity was observed. The metabolic activity was even further decreased after co-treatment with Lapatinib. Lapatinib targets the HER2 protein and is clinically used in combination therapy to treat advanced breast cancer. Novel approaches to target gene expression might thus improve the effect of conventional protein targeting drug treatment regiments. In conclusion, the induction of repressive histone marks by epigenetic editors can result in gene expression modulation. Since epigenetic marks have the potential of being mitotically stable, this study describes a powerful approach to efficiently silence (onco)genes. Epigenetic editing of the PLOD2 promoter in a fibrotic setting To further explore the application of epigenetic editing for downregulation of gene expression, we set out to decrease PLOD2 expression by targeted DNA methylation (Chapter 8). So far, this thesis has focused on several cancer types, but here we further explore the multiple facets of ATF-technology in a fibrotic setting. PLOD2 is a key gene involved in undegradable pathological scarring and its expression is strongly increased in fibrotic conditions mediated predominantly by TGFβ1. In that respect, downregulation of PLOD2 in fibrotic conditions may decrease the detrimental effects of fibrosis, making deposited collagen degradable and resolve fibrosis. First, we screened eight PLOD2-specific ATFs for their ability to downregulate PLOD2 expression. From the eight engineered ATFs, two ATFs were able to significantly downregulate PLOD2 expression, up to 99,5% in human fibroblasts using the repressive SKD domain. Also under profibrotic conditions, the two ATFs substantially prevented PLOD2 induction. To achieve sustainable downregulation of PLOD2, the PLOD2-targeting ZFPs were connected to CpG Methyltransferase (M.Sss1) and transiently expressed in human fibroblasts. We were able to successfully methylate the PLOD2. Ten days after the TFGβ1-induced activation of the fibroblasts, PLOD2 expression was still decreased, demonstrating the sustainability of the epigenetic editing approach. However, also SKD-mediated downregulation was sustained after ten days, even stronger compared to M.Sss1. Therefore, we analyzed if the SKD-constructs affected the epigenetic state of the

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PLOD2 promoter. And indeed we found that SKD-mediated downregulation overwrites epigenetic marks, as we previously also observed for HER2-SKD-ATFs. While control conditions showed the abundantly present H3K4me3, AcH3 and AcH4 activation marks, the ATF-mediated repression showed a sharp reduction of these marks, while enhancing the presence of the repressive histone marks H3K9Me3 and H3K27Me3. These epigenetic changes were most likely responsible for the ‘inheritance’-effect. As the PLOD2-targeting constructs could downregulate PLOD2 under profibrotic conditions, they may be excellent tools to serve as treatment for fibrotic conditions, such as dupuytren’s disease. Previously, long-term repression was also observed upon targeting of DNA methyltransferase 3a to the Maspin and Sox2 promoter (41). Apperantly, stable reprogramming by rewriting the DNA methylation code offers a very potent avenue to permanently decrease gene-expression. For M.Sss1, this is the first report that described the targeted methylation of an endogenous promoter in mammalian cells. Previously, M.Sss1 was used to methylate endogenous targets in the context of yeast chromatin (54). Future research may determine the most efficient methylase for rewriting epigenetic landscapes. As downregulation through epigenetic editing may be applicable for any gene, this approach has potentials in many diseases to silence genes. Future Work/Perspectives ATF-technology offers a great tool for gene expression modulation and for studying the functional consequences. This thesis demonstrated the many potentials of ATFs. Although gene-expression modulation can also be achieved using traditionally methods such as cDNA overexpression or siRNA-mediated downregulation, ATFs posses important advantages as discussed in the introduction. Since the first published ZF-ATF in 1998 (55), many groups have reported successful gene expression modulation by ATFs (56), mostly aimed to improve pathological conditions in the studied disease. However, the performed studies are mostly limited to pre-clinical models, and the future goal should be to make ATF treatment effective and safe for clinical use. Nevertheless, several clinical trials using ZFPs have been performed (by Sangamos Bioscience) with to some extent promising results. The only ATF-trials involved the treatment of diabetic neuropathy by targeted modulation of VEGFA. Although ATF-therapy proved safe, no beneficial effects were observed compared to placebo (57), which was partly explained by the unexpected improvement in general health conditions of the placebos. Another ZFP-trial, which is still ongoing, aims to develop a "functional cure" for HIV/AIDS. In this trial, ZFPs were linked to a nuclease (ZFN) for genetic editing purposes. The designed ZFN disrupts CDR5 on CD4 cells (58), making it difficult for HIV to infect the cell. So far, this trial has given promising results, as the ZFN were able to be sufficiently expressed in target cells, resulting

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in a significant decrease in viral load in HIV patients. In this trial, the constructs were delivered by an adenoviral vector, which may have greatly enhanced the delivery of the constructs to the target cells compared to the VEGFA trial using plasmid delivery. Maybe, this delivery approach would also benefit ATF-mediated effects in clinical trials. Another advantage of adenovirus based delivery, compared to for example retroviral based delivery used in this thesis, is that no hazardous viral DNA/RNA integrate into the host DNA (59), which makes this system more safe for clinical use. The suitability of gene therapy is highlighted by the recent approval of the first viral vector (adeno-associated virus) based therapy, were lipoprotein lipase deficiency is treated (60). Advances in such delivery systems are very beneficial for developing safe therapeutic strategies utilising ATFs. For cancer, however, specific delivery of ATFs towards the tumor faces many challenges. Systematic delivery of ATFs by an adenovirus leads to accumulation of the viral vector in the liver (61) and therefore a targeted approach is needed. A potential strategy for specific delivery of the ATF (or ATF carrying vector) to tumor cells may be the encapsulation of the ATF in a vesicle, which is directed to the organ of interest by tumor specific antibodies. The ATF-carrying vesicles might be specifically directed to cancer cells through coverage with highly specific antibodies which only recognize cancer cells. This strategy would use a double targeting approach; first the organ of interest is targeted and subsequently the gene of interest. The feasibility of this approach is highlighted by the successful delivery of encapsulated siRNA towards target tissue, leading to successful downregulation of target gene-expression (62). Next to adenoviral delivery, the direct delivery of the ATFs as proteins may be another promising strategy to deliver ATFs. In a recent paper, direct protein delivery has been successfully explored for gene manipulation in vivo (63). The advantage of protein delivery is that the ATF is delivered directly and does not require protein synthesis from DNA strands which can be inefficient, while also the chance for rearrangements after transduction are reduced. As with adenoviral therapy, easy accessible organs or cell types may benefit more effectively from protein delivery (blood cells, limbs). For example, patients suffer from dupuytrent’s disease have impaired functioning of the hand probably caused by dysregulation of processes in the connective tissue of the affected area. The connective tissue is readily accessible by ATF-infusion, which may normalize the abnormal gene-expression patterns. As PLOD2 dysregulation may be an important factor for this disease, treatment with the PLOD2 regulating ATFs may prevent progression of this disease. Future efforts will determine if ZF-based ATFs will become suitable for clinical application, as also other epigenetic therapies have been FDA approved. For now, ATF-technology for clinical use is patented by Sangamo research, which makes it commercially uninteresting for many parties to study the clinical applications of ATFs.

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This thesis demonstrated the potentials of ATF technology to normalize gene expression in disease. However, healthy cells may also benefit from gene expression manipulation to better withstand the constant treats certain cells are constantly exposed to. For example, increased NRF2 expression, the master regulator of many antioxidants, may protect healthy ovarian cells from oxidative stress induced DNA damage during menstruation (64). We have designed and engineered NRF2-targeting ATFs, which were able to successfully modulate NRF2 levels on RNA and protein level. The upregulated NRF2 expression seemed to protect healthy ovarian cells from oxidative stress (65). With such progresses, ATFs may be used to prevent all kinds of diseases in the far future. In the last years novel strategies other than ZFP-technology have been developed to target the DNA for gene expression modulation or (epi)genetic editing. In that respect, transcription activator-like effector (TALE) proteins have been presented as an alternative promising higher success with regard to specificity and predictability (30, 66). TALEs are DNA binding proteins from the bacteria genus Xanthomonas, which are composed of modular repeats of 33-35 amino acids that target a single base pair. Since the DNA specificity of TALEs was decoded in 2009 (67), many groups have successfully designed TALEs for gene-targeting. The main disadvantage of TALEs may be their large size, which complicates delivery into cells for clinical purposes, but also limits their accessibility to hypermethylated promoters (68). However, one study compared the differences between ZFPs and TALEs for their ability to decrease DNA methylation levels at a certain target site when fused to Tet1, but they did not find differences between the two targeting platforms (21). Interestingly, they observed similar effects on CpG methylation after targeting Tet1, as we observed for targeting Tet2 to the hypermethylated C13ORF18 and CCNA1 promoter. The lack of differences between the two targeting systems may be caused by the large size of Tet-demethylases fused to ZFPs or TALEs; the advantage of ZFPs compared to TALEs, that they are small molecules, is not valid anymore. Future studies may give more clues about the ideal gene-targeting platform, which may ultimately be gene-dependent. Recently, yet another strategy for the targeting of endogenous genes has been reported called the clustered, regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system (29). This approach has been employed from the immune function in archaea and bacteria and relies on the targeting of genes by guided RNA molecules. In archaea and bacteria, the RNA-guided Cas9 proteins facilitate sequence-specific cleavage of pathogenic DNA (69). The RNA-guidence molecules can be easily redesigned to target any sequence of interest. In contrast to ZFPs and TALEs, which depend on an artificial protein-DNA code, the nucleotide-directed specificity of the CRISPR/Cas9 is believed to be more convenient and easier to use (70). The targeting of genes only requires RNA synthesis or PCR amplification, other than TALEs and ZFP-based technology which

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needs to be customized for every new target, and the use of these technologies requires specific expertise. Although, each of these three systems has and will contribute greatly to the advances in medical research, future efforts will determine any therapeutic potential for these gene-targeting approaches, as was already shown for ZFPs in the clinical trial. With respect to epigenetic editing -the targeting of epigenetic enzymes towards promoter regions to obtain sustainable expression modulation- we could downregulate a target gene (PLOD2) for at least ten days. Whether target genes can be ‘permanently’ repressed should be addressed in future studies. Prolonged re-expression was achieved using ATFs, co-treated with epigenetic drugs for the measured five days. In the Tet1 study by Maeder et al (21), re-expression was achieved up to ~20 days. However, seven days after expressing the Tet-fusions, approximately half of the induced gene expression was lost for the measured three target genes. After 14 days, expression was almost returned to the original level for these genes. In contrast, PLOD2 levels were stably downregulated for the measured ten days, with no measureable increase of PLOD2 expression during this period. The induced silencing of PLOD2 indicates that our hit-and-run strategy may have led to complete reprogramming of the epigenetic state of the PLOD2 promoter, as also indicated by the DNA and histone methylation patterns after the targeted intervention. To achieve permanent re-expression by targeted demethylation may be more challenging compared to downregulation, as CpG remethylation is likely to occur (21). Efficient DNA demethylation of target genes is of key importance to achieve the sustained re-expression. The described methods and observations about targeting demethylases to biomarker genes in this thesis may help to achieve this, and this is now subject of further investigation. Future efforts in unravelling the elusive DNA demethylation pathway may give more clues to increase the efficiency of targeted demethylation. Possible strategies to prevent remethylation, e.g. co-treatment of demethylases with TSA or knock-down of methyltransereases (by targeted methylation), may also further improve sustainability. In the future, the combined actions of ATFs, epigenetic editors and epigenetic drugs may eventually result in permanent re-activation of target genes to gain full control over the transcriptome. In conclusion, this thesis described a powerful approach to specific regulate expression at the single gene level to combat disease. Moreover, the established protocols (for epigenetic reprogramming) enable scientists to ‘permanently’ silence or re-activate any desired target gene (or other RNA-coding sequences (e.g. long non-coding RNAs)) with potential single gene resolution of any chosen gene. Furthermore, the described rewriting of epigenetic marks contributed to unraveling nature’s rules in stable gene regulation. The ultimate goal would be to achieve sustained reprogramming in vivo.

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