Opportunities and Challenges of Epigenetic Editing in Human Diseases
Goubert, Désirée
DOI:
10.33612/diss.173201281
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Publication date: 2021
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Goubert, D. (2021). Opportunities and Challenges of Epigenetic Editing in Human Diseases: Towards the Curable Genome. University of Groningen. https://doi.org/10.33612/diss.173201281
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General Discussion and
Future Perspectives
In many human diseases epigenetic regulation of genes goes wrong (1-4), which can have severe consequences on gene expression and cellular behaviour as well as on sensitivity to treatment and development of resistance (5, 6). The research presented in this thesis focusses on identifying and reversing epigenetic malfunctions that are associated with disturbed gene expression patterns in human diseases. The overall aim was to use Epigenetic Editing to reprogram specific target genes by rewriting epigenetic marks, without changing the primary DNA sequence (Chapter 1).
In Chapter 2 the scope of the EpiPredict consortium, of which this PhD project was part, is
described in detail. The overall focus within EpiPredict was to uncover the role of epigenetic regulation in the development of resistance to endocrine therapy in oestrogen receptor positive (ER+) breast cancer. A systems medicine approach was employed by combining multidisciplinary research strategies and next generation technologies (epigenetic/genetic profiling, protein pathway activation, metabolic pathway profiling, gene-specific epigenetic interference technologies and computational approaches). Results from these combined efforts include detailed procedures and protocols describing powerful tools (Single-Molecule RNA FISH, creation of dCas9-expressing cells lines, Chromatin Immunoprecipitation and High-Throughput Sequencing) which can be used to study epigenetic regulation (7-9). Profiling studies within the consortium provided us with valuable target genes, involved in the development of endocrine therapy resistance such as HES1, ESR1 (10), YY1 (6) and CD44 (11). Several sgRNAs were developed and tested for these genes (Figure 1). The sgRNAs designed to target ESR1 were based on previous studies describing successful targeting of Zinc Finger (ZF) proteins that were designed in our laboratory (12). ZF proteins are naturally occurring transcription factors which are, besides CRISPR-dCas9 sgRNAs, used as a programmable DNA targeting platform to guide effector domains to specific target genes (Chapter 3). Both the
artificial transcriptional repressor SKD (not possessing enzymatic activity but recruiting other epigenetics enzymes), as well as targeted methylation by variants of the DNA methyltransferase M.SssI were able to downregulate the expression of ESR1 in MCF-7 breast cancer cells, but not in ovarian cancer SKOV3 cells using our designed ZFs (12). The observed downregulation of ESR1 could not be repeated using sgRNAs together with CRISPR-dCas9 coupled to any artificial transcription factor (ATF) or to an epigenetic enzyme, reflecting the differences between the targeting platforms and the experimental set ups: The previously
used ZFs were stably expressed in the cells and thus were constantly present, whilst the sgRNAs were transiently transfected (to be able to assess long-term effects of the constructs, even when they are no longer expressed in the cells). Further, the relatively small size of ZF proteins (13, 14) compared to the larger size of CRISPR-dCas9 (approximately 8 times larger than ZFs) could explain the difference in e.g. gaining DNA accessibility. Issues of delivery, sgRNA efficiency and context-dependency are described in detail in this Chapter.
Due to lack of consistent repressive or inducible effects, or high variability of the effect on specifically the CD44 gene we did not continue to study HES1, ESR1, YY1 or CD44. Yet, the possibility to predict and avoid resistance to endocrine therapy will be a step closer to a so-called tailored therapy for breast cancer patients. Such epigenetic insights could be translated into diagnostic or prognostic tools to differentiate patients for their likelihood to develop endocrine resistance and predict the efficiency of additional drugs to counteract resistance in high risk patients.
To this end, the CD44 sgRNAs are currently further investigated in DKFZ, as this target is known to be associated with breast cancer invasiveness and stemness (15) and therefore might be important for endocrine resistance acquisition. Recent findings indicate an increase in tumour cells expressing high CD44 levels under oestrogen deprived conditions, which mimics a resistant environment (11). Early findings from DKFZ (16), under supervision of Prof. Dr. Stefan Wiemann, show that re-sensitization of resistant cells could be achieved using CRISPR tools. In long-term oestrogen deprived (LTED) MCF-7 cells (mimicking a resistant breast cancer phenotype) stably expressing a fusion of dCas9 and a histone methyltransferase (G9A), the targeting of CD44 with the sgRNAs designed in our lab resulted in repression of
CD44. The treated cells were more sensitive to oestrogen–deprived conditions, leading to a
significantly decreased proliferation compared to control cells which were transfected with a non-targeting control sgRNA. This finding strongly suggests the involvement of CD44 in the maintenance of endocrine therapy resistance in MCF-7 LTED cell lines.
Figure 9: Schematic representation of target gene promoter regions and locations of the respective sgRNAs (Green
arrows) and Zinc Finger Proteins (Blue arrows). CG islands (CGI) are represented by the red bar and the locations of the Transcription Start Site (TSS) is depicted.
We further focussed on using Epigenetic Editing in other target genes with known roles in cancer, as well as in fibrosis and airway diseases, including PLAU (Chapter 4), PLOD2 (Chapter 5), and UCHL1 (Chapter 6).
Achieving functional effects using Epigenetic Editing
Increased levels of PLAU have been associated with a worse prognosis and an increase in aggressiveness, metastasis, and invasion of breast cancer (17-22). By upregulating PLAU in non-invasive, hormone-sensitive, oestrogen receptor α positive luminal A MCF-7 breast cancer cells, using the artificial transcriptional activator dCas9-VP64, the oncogenic activity of this gene was indeed confirmed (Chapter 4). We observed an increased migration of breast
cancer cells with an increased PLAU expression, indicating a more aggressive phenotype. This is in line with other reports which describe induction of PLAU using untargeted approaches in other cell lines (23-30). These reports make use of PLAU activating compounds that also affect other genes which could lead to unwanted side effects in a clinical setting. In some of these reports, the increased migratory capacity after PLAU upregulation was successful in promoting wound healing (27, 30), indicating a potential application of PLAU induced upregulation. Our targeted PLAU-induction platform using dCas9-VP64 could thus be of use in pathological conditions such as ischemic brain injury (31-33), lung fibrosis (34), male infertility (35), type 2 diabetes mellitus (36) and diabetic keratopathy (37).
Next, we set out to repress PLAU in hormone-insensitive, triple negative MDA-MB-231 breast cancer cells to inhibit the migration potential of these aggressive cells. No significant downregulation by using the non-catalytic KRAB repressor (SKD), nor targeted DNA or histone methylation, using the DNA methyltransferase M.SssI or the histone 3 lysine 9 methyltransferase G9A, could be accomplished at this point. This is most likely because of poor delivery of our constructs. At the beginning of this project we were well aware of the low transfection efficiency in MDA-MB-231 cells, as is also shown by others (38-40). To overcome this problem, we created stable cells expressing dCas9- effector domains (EDs), a procedure that is described in detail in Chapter 7. Cell lines stably expressing dCas9-EDs are
expected to improve screening approaches, as dCas9 plasmids are relatively large making efficient delivery using transient transfections more difficult (8, 41). Several cell lines have
been transformed in our lab into stable cells continuously expressing several dCas9-EDs, however we were not able to show improved transient transfection efficiency compared to wild-type cells. For example, a more efficient repression of PLOD2 was observed in Chapter 5
using double transient transfection (plasmids expressing dCas9-SKD and sgRNAs) in wild-type HEK293T cells as compared to repression of PLOD2 in stable dCas9-SKD expressing HEK293T cells. Also stable expression of dCas9-VP64 in BEAS-2B cells in Chapter 6 did not significantly
improve on the UCHL1 induction achieved with transient transfection of this ED, despite the 100 times higher expression of dCas9 in stable cells compared to transiently transfected cells. Despite increasing the amount of sgRNA transfected in an attempt to improve upon our induced effects, we did not see a consistently higher gene induction or repression in any of the other genes tested in the stable cells. This effect could illustrate that the maximum effect of induction or repression of gene activity that can be achieved using transient transfection has already been reached in wild-type cells.
Since lentiviral transduction efficiency is known to be much higher than transient transfection (42-44), we set out to transduce MDA-MB-231 cells stably expressing dCas9-EDs a second time with lentiviral sgRNAs targeted to the PLAU promoter. No significant repression was observed, even though a trend towards downregulation was present in cells expressing the DNA methyltransferase Q147L or the histone 3 lysine 9 methyltransferase G9A. M.SssI-Q147L is a mutated variant of the CG-specific prokaryotic DNA methyltransferase M.SssI which has ~10% activity of the wild type M.SssI, the catalytically inactive mutant is M.SssI-E186A, which is used as a control. The lower DNA binding affinity of Q147L has been proven to increase the specificity of targeted DNA methylation over the wildtype M.SssI (45). In other cellular contexts and for other genes, these EDs did show repressive effects, which already shows some of the chromatin context-dependency that affects the efficiency of Epigenetic Editing, which is explained in more depth later in this Chapter. For example dCas9-G9A, but not it’s catalytic mutant, was able to achieve mitotically stable silencing of SPDEF in the type II alveolar carcinoma A549 cell lines as well as in MCF-7 cells (46) and M.SssI-Q147L, but not it’s catalytically inactive mutant (dCas9-E186A), induced gene repression of PLOD2 in fibroblasts, human embryonic kidney cells (HEK293T) and MDA-MB-231 breast cancer cells, but not in MCF-7 cells (Chapter 5).
In a second attempt to repress PLAU in MDA-MB-231 cells, we used Nucleofection® electroporation to transfect MDA-MB-231 dCas9-ED stable cells to increase the efficiency of the sgRNA delivery. This physical form of transfection employs an electrical force on the cells to create transient pores in the cell membrane, enhancing the uptake of molecular tools such as Epigenetic Editing constructs. Using this method, many promising in vivo gene editing applications are already being developed in mice (47-51). Because this method can be quite toxic for cells, it would be mainly suitable for ex vivo applications in humans, in which patient cells are taken out of the body, treated, and put back into the patient, as is shown by ongoing clinical trials using this physical form of transfection (52-55). Unfortunately, we did not see any effect on PLAU expression in any of the cell lines transfected by Nucleofection electroporation. In our Nucleofection experiments, we transfected three different fluorescently-tagged plasmids: a GFP (green fluorescence) -tagged control plasmid, a GFP-tagged sgRNA plasmid (empty, not targeted towards any gene) and a mCherry (red fluorescence) -tagged dCas9-NED (No Effector Domain). When cells successfully take up these plasmids, the fluorescent signals can be measured and efficiency of transfection can be determined. Although the transfection efficiency of cells Nucleofected with the small GFP control plasmid was sufficiently high (±60-70%), no fluorescent signal could be observed for the other two plasmids, despite using various concentrations of plasmid and different Nucleofection programs. Nucleofection of HEK293T with the same three plasmids showed similar results, with only very weak fluorescent signals being visible from the GFP-tagged sgRNA plasmid and the mCherry-tagged dCas9-NED plasmid. This suggests both plasmids were not properly transcribed within the cells. Future research should focus on optimizing delivery of targeted Epigenetic Editing tools to change gene expression and subsequently achieve changes in cell functioning.
Delivery of Epigenetic Editing constructs
One promising virus-free delivery method that could be further investigated for this purpose is the use of ligand-directed targeting approaches which target e.g. cancer cell specific surface receptors. These approaches, which might have therapeutic potential, make use of liposomes and other nanoparticles (56-58). Because of this, the therapeutic tools are more efficiently taken up by the cells, and are protected from degradation by the biological environment (56).
This method is not limited to only targeting cancer cells and holds the possibility to selectively target specific tissues (59, 60). These methods are attractive for clinical use as uptake by patients is improved compared to e.g. virus-based delivery methods (61). However, a possible disadvantage is the shorter in vivo life-time some of these extracellular vesicles can have compared to adeno-associated virus (AAV) -mediated delivery (60, 62). Also, for CRISPR-dCas9 based approaches such novel delivery techniques are showing promise for in vivo gene activation. Kretzmann et al. used dendritic polymers in a targeted intravenous approach to reactivate the tumour suppressor genes MASPIN and CCN6 in a sustained and efficient way in a mouse model of breast cancer (63). This reactivation led to an efficient and long-term cancer growth inhibition with negligible toxicity, outlining a targeted and effective method with potential to treat aggressive malignancies. Specifically in the difficult to transfect MDA-MB-231 cells an ultrasound targeted microbubble destruction (UTMD) technology has recently been optimized to deliver constructs in these cells (40). Here, Zhang et al. make use of the mechanical effects of ultrasound, combined with acoustically responsive microbubbles or droplets, creating transient pores in the cell membrane which enhances introduction of molecular tools. This technology has already shown great promise for the delivery of drugs or genes in models of solid tumours (64, 65), obesity (66) and cardiovascular disease (67). The most promising in vitro delivery system for Epigenetic Editing tools at the moment is adeno-associated viral (AAV) delivery, which is also the most commonly used vector for packaging and delivery of CRISPR components in vivo (68-72). For in vitro based research, lentiviral delivery is equally efficient, however upon infection lentiviruses randomly integrate into the genome of the host cell, leading to stable expression of the Epigenetic Editing tool. The adeno-associated virus is a non-pathogenic virus with a very mild immune response capable of delivering constructs in a tissue-specific way, with a high efficiency and without integration into the host genome, which could leads to harmful side-effects (73-75). Despite these wonderful properties, only very few Epigenetic Editing studies have successfully delivered CRISPR-dCas9-EDs in vivo using AAV delivery. This can be attributed to the limited packaging capacity of these AAV vectors (~5 kb) and the large size of the Epigenetic Editing constructs (76-78). Ongoing improvements such as smaller dCas9 orthologues and a split dCas9 system (in which the dCa9 enzyme is split, one part will be coupled to the ED, the other to the sgRNAs and the platform will only be active when both components bind) will
ultimately decide the success of AAV delivery of Epigenetic Editing constructs in a clinical setting (79).
Technical improvements
In view of the many possibilities Epigenetic Editing holds, technical improvements have been assessed in this thesis. For example in Chapter 6, we tested different experimental strategies
to modulate the gene expression of UCHL1, a gene with relevance in cancer and airway diseases. One such an improvement is the insertion of additional RNA hairpins into the sgRNA plasmid to form the sgRNA2.0 system (80). These RNA hairpins are recognised by RNA-specific binding proteins, which can be coupled to the same type of effector domains as to CRISPR-dCas9. The sgRNA2.0 is thus capable of recruiting both the RNA-specific binding protein effectors as well as the CRISPR-dCas9-ED, which can lead to amplification of the efficacy of induced effects (80-82). For example, Konermann et al. showed that targeting of sgRNA2.0 to
Neurog2 with a combination of both MS2-VP64 and dCas9-VP64 resulted in an additive effect,
leading to 12-fold higher increase compared to targeting only dCas9-VP64 (80). In our two cell lines however, MS2-targeting of p65-HSF1 (a transcriptional activator) did not improve the VP64-induction of UCHL1 gene expression even though the MS2-tagged p65-HSF1 on its own did induce gene expression up to 4-fold. This could indicate that either the maximum level of induction is reached in these cells, or and more likely, the lack of proper controls to ensure efficient delivery of the constructs (e.g. fluorescent tags to sort successfully transfected cells). To avoid the current difficulties of efficient delivery of large or multiple components, researchers can turn to methods of selection such as antibiotic resistance assays and fluorescence activated cell sorting (FACS) to enrich the transfected cell population (83). FACS can be performed upon delivery of fluorescent tags such as GFP or mCherry fused to the (d)Cas9 (84-86), to the sgRNAs (87), or to both allowing dual fluorescent tag sorting (88, 89). Cells that have successfully taken up the fluorescently-tagged constructs, will emit fluorescence at a specific wavelength when they are excited by a laser, after which they can be isolated from the bulk population and further investigated (90, 91). FACS, in our case, was the most successful improvement that we tested to increase upon the induction of UCHL1 using CRISPR-dCas9 in Chapter 6. The FACS sorting procedure utilizing indirect dCas9-mCherry
fusions resulted in an increased efficiency to measure gene expression changes in transfected
cells, displaying a 6- and 15-fold induced UCHL1 expression for the lung cancer H1299 and lung epithelial BEAS-2B cell lines, respectively, compared to the 2.2- and 2.8-fold increase observed in bulk cells, that were not sorted. In Chapter 4, no fluorescent signal could be
observed after nucleofection of the larger GFP-tagged sgRNA plasmid, or mCherry-tagged dCas9-NED, however this is likely because the constructs were not successfully delivered into MDA-MB-231 cells. Several fluorescently-tagged CRISPR-dCas9-ED have been created by us
(Chapters 4 and 6) that can be used to increase the population of successfully transfected
cells using FACS.
Sustainability of Epigenetic Editing
Besides delivery, another important challenge to overcome before epigenetic reprogramming can be developed into a straightforward technology for effective and specific interventions, is the potential dependency on genomic context to enable sustained transcription modulation. Sustained transcriptional modulation has been achieved in several in vivo models (71, 92, 93), for example, AAV delivery of dCas9-KRAB targeting Pcsk9 (a regulator of cholesterol levels) in liver cells of adult mice, resulted in reduction of Pcsk9 gene expression and reduction of cholesterol levels for a period of at least 24 weeks (71). Xenograft studies, in which NUDE mice were injected with tumour cells that stably express either a DNA methyltransferase or empty vector demonstrated stable SOX2 repression with maintenance of DNA methylation and long-term breast tumour growth inhibition, which lasted for more than 100 days after implantation of the tumour cells (92). Reactivation of Sim1 and Mc4r in haploinsufficient mice (in which loss-of-function mutations in one gene copy (as opposed to two) can lead to reduced amounts of protein and, consequently, disease) using AAV delivery of dCas9-VP64 into the hypothalamus of these mice led to reversal of the obesity phenotype normally seen in mice lacking expression of these genes, which was still observed nine months after injection of the constructs (93).
All the above examples, however, make use of the episomally maintained AAV or stably engineered cells, not reflecting true epigenetic reprogramming. The current consensus holds that to achieve sustained effects by epigenetic reprogramming, multiple effector domains (e.g. KRAB, DNA methyltransferase and/or histone modifiers) are required (75, 94-96). Transient expression of combinations of these effector domains resulted in synergistic effects
and long-term repression of several genes by deposition of repressive histone marks and de
novo DNA methylation. For clinical applications however, the requirement of multiple
components would be a serious limitation. In Chapter 5, we thus tested whether the fibrosis-
and cancer-associated gene PLOD2 can be repressed in a sustained manner by the individual effector domains DNA methyltransferase M.SssI, or the non-catalytic KRAB repressor (SKD). Both effector domains when fused to PLOD2-targeting ZFs, were able to repress PLOD2 also after 10 days of TGFβ1 stimulation (which induces PLOD2 expression in fibrosis (97-99)). To ensure the expression of the constructs in all analysed cells, the ZF-fusions were stably expressed by fibroblasts and MDA-MB-231 cells under the control of a doxycycline-inducible expression system. This entails that the expression of the ZFs is induced by adding doxycycline to the medium in which the cells grow. However, leakiness is a frequently observed phenomenon of this system. We indeed found similar levels of PLOD2 repression in cells with or without doxycycline, preventing us from making conclusions on the sustainability of SKD- or M.SssI-induced effects on PLOD2 expression using this system. We therefore investigated sustainability of the effects using the transient sgRNA expression system in HEK293T cells stably expressing dCas9-SKD or dCas9-M.SssI variants. Upon targeting PLOD2 in engineered HEK293T cells, SKD led to a sustained repression (after 2 and 12 days), while repression induced by M.SssI seems to take a longer time (only detectable after 12 days). This was a very striking observation, especially given that the current paradigm of SKD induced repression holds that transcriptional repressive effects are transient in somatic and cancer cells (75, 94, 96, 100-104). Using KRAB as an effector domain, Thakore et al., showed silencing of a cholesterol regulating gene (Pcsk9) in the liver of adult mice for a duration of at least 24 weeks upon delivery using AAV vectors (71). However, as mentioned before, these delivery vectors are episomally maintained, preventing conclusions from being drawn about the mitotic stability of the effect of KRAB itself. We therefore set out to confirm in a truly transient system, the sustainability of SKD-induced PLOD2 repression, by transfecting the plasmids to express dCas9-SKD as well as the sgRNAs into wildtype HEK293T cells. This resulted not only in a sustained repression, as previously seen in stable dCas9-SKD-expressing cells, the repression was also more efficient using double transient transfection (plasmids expressing dCas9-SKD and sgRNAs) as compared to repression of PLOD2 in stable dCas9-SKD expressing cells. Using the truly transient (double-transfection) CRISPR-dCas9 system, we showed that SKD as well as M.SssI targeting is sufficient to induce sustained PLOD2 repression. Targeted
transcriptional modulation using either M.SssI or SKD thus has the potential to evolve into anti-PLOD2 therapeutics against fibrosis.
Chromatin context-dependency of Epigenetic Editing
A critical hurdle to overcome before Epigenetic Editing can be implemented into the clinic is the gene and chromatin dependent optimization that is required, as targeting effects seem to be rather context-dependent. This context-dependency refers to several aspects including the local chromatin state and efficiency of sgRNAs (109, 110), the effect of the targeted ED or the edited epigenetic mark (111) and transcriptional state of the target gene (112). The chromatin state refers to the way DNA is modified and packed into the nucleus, and this influences the efficiency of DNA-targeting constructs, including CRISPR-dCas9, as well as their intended outcome (efficient localisation of EDs and successful deposition of epigenetic marks at the intended site does not always lead to the same functional effect) (113-116).
The chromatin context-dependency is already apparent in the first steps of the Epigenetic Editing approach i.e. the design of the DNA-binding platform to target genes of interest. For example, in Chapter 5, designing a different set of sgRNAs, which target the DNA at a
sequence similar to ZFs that were shown to successfully repress PLOD2 expression, did not lead to any repression in HEK293T cells stably expressing dCas9-SKD. Also in Chapter 6 we
could not improve the 5-fold induction in gene expression that was initially achieved by targeting dCas9-VP64 to UCHL1, by designing and combining sgRNAs that bind other genomic sequences. In MCF-7 cells (low PLAU expression), using the same PLAU targeting sgRNAs lead to significant induction of gene expression, whilst no significant repression could be achieved in MDA-MB-231 cells (high PLAU expression) (Chapter 4). As sgRNA design software does not
take the different chromatin contexts in different cell models into account, nor the type of effector domain that is targeted, the sgRNA design rules could be improved based on more systematic screening efforts (114, 117-119).
The effect of a targeted effector domain is also influenced by this context-dependency. For example, a combination of PRDM9 and DOT1L targeted to PLOD2 in C33A cervical cancer cells resulted in a significant and sustained upregulation (111). Using the same effector domains,
separately or in combination, did not lead to a short-term or long-term induction of PLAU in MCF-7 breast cancer cells (Chapter 4), whilst induction by dCas9-VP64 used in the same set
of experiments, as a positive control, did give a significant transient induction in MCF-7 cells. Transfection of only dCas9-SKD together with PLOD2 targeting sgRNAs gave a sustained repression of PLOD2 in HEK293T human embryonic kidney cells and MCF-7 cells, but no sustained repression was observed when SKD was targeted to another gene (SPDEF) in MCF-7 cells (Chapter 5). Co-transfection of dCas9-M.SssI-Q147L with dCas9-SKD, but not either of
these EDs individually, lead to a significant and sustained repression of UCHL1 in HEK293T, but not H1299 lung cancer cells (Chapter 6). Targeting methylated or unmethylated sites with
dCas9-VP64 has been shown to result in a difference in the efficiency of transcriptional upregulation of a gene (111). Hypermethylated genes ICAM1 and RASSF1a were not upregulated by targeting dCas9-VP64, whilst unmethylated genes EpCAM and PLOD2 did show a significant upregulation (111). This might suggest that dCas9-VP64 is not able to access the promoters of hypermethylated genes, explaining the lack of effect upon its targeting. Finally, the effect of epigenetic marks and whether or not they are maintained can also be chromatin context-dependent, so whilst an ED can lead to the deposition of epigenetic marks on a given target gene, the functional outcome can be different in e.g. different cell types. For example, efficient PLOD2 DNA methylation was detected in fibroblasts expressing either ZF7-M.SssI or ZF8-ZF7-M.SssI, even though at day 10 gene expression repressive effects of ZF8-ZF7-M.SssI were lost (Chapter 5). However, the difference in efficiency of induced DNA methylation
(63.4% induced by ZF7-M.SssI compared to 48.7% methylation induced by ZF8-M.SssI) could also explain the loss of PLOD2 repression seen in the latter. This could imply that to achieve sustained repression of the PLOD2 gene by targeting the M.SssI enzyme, a threshold amount of DNA methylation has to be induced and/or CpGs critical for transcription initiation need to be methylated. Another explanation could be that the target location of ZF7 was more responsive to deposited DNA methylation and subsequently lead to gene repression. Furthermore, in fibroblasts targeting of ZF7-M.SssI increased the presence of repressive histone marks H3K9me3 and H3K27me3, whilst this was not the case upon targeting ZF7-M.SssI in cancer cells (Chapter 5). This could be due to differences in growth kinetics of the
cells, which is much faster in the cancer cells compared to fibroblasts, or by the continuous TGFß1 stimulation applied to the fibroblasts but not to the cancer cells. Another example of context-dependent epigenetic marks is shown in Chapter 6. Here, an efficient UCHL1 DNA
methylation of 70% in HEK293T cells, using a combination of M.SssI-Q147L and dCas9-SKD, lead to a significant 40% gene repression in the sorted cell population. An efficient methylation of 60% in H1299 cells however, did not lead to a repressive effect, despite the fact that a region was targeted where DNA methylation is generally associated with UCHL1 repression (120) (Chapter 6). Similarly, sustained repression was achieved using targeted DNA
methylation in some studies (92, 121-123), but this could not be confirmed for other targeted genes (87, 124). These examples show that also the maintenance of epigenetic reprogramming is chromatin context-dependent.
Genetic vs. Epigenetic Editing
While, besides ex vivo, now also the first test of in-body (in vivo) gene editing in humans has shown encouraging results, one might be wondering what the added advantage of Epigenetic Editing is over the more developed application of gene editing. Early results of these in vivo gene editing trials suggest that the cells of the patients were successfully modified and that the that gene editing is safe in humans (125-127). Gene editing can correct errors in the genome by changing the DNA sequence, whilst Epigenetic Editing changes the expression of genes, possibly with sustained effects, without changing the DNA sequence. Ongoing clinical trials can be registered on https://clinicaltrials.gov/, where they get an unique identification or NTC number, and as of 2020 a handful of gene editing trials have entered clinical trials. For example, researchers from Sangamo Therapeutics are using gene editing techniques to insert a healthy copy of the iduronate-2-sulfatase gene into liver cells, and in this way treat people with Hunter syndrome a condition in which patients are not able to break down complex sugars (NCT03041324) (128, 129). At the same time, clinical trials are also ongoing to treat hemophilia B (NCT02695160) and Hurler syndrome (NCT02702115). In all these three in vivo cases, ZFs are targeted toward the disease-causing gene and delivered into the patients via virus based approaches. For clinical applications, despite a difficult start, viral vectors (Adeno Associated Viruses and Lentiviruses) are reaching widespread acceptance (130). Recently, other researchers have also tested CRISPR-based gene editing inside a person’s body for the first time. Allergan and Editas Medicine treated the first patient with Leber congenital amaurosis 10, an inherited type of blindness caused by mutations in the CEP290 gene (NCT03655678). Microscopic droplets carrying an inactivated virus are administered by a
subretinal injection in one eye. Results are not yet known, and the primary goal of this early-stage trial is to test the safety of the approach, which is partly why only one eye is treated initially (131).
Despite these impressive and very important steps forward, gene editing techniques also pose several important downsides that could limit their clinical potential. Given the fact that DNA alterations are permanent, one of the most critical considerations is the specificity of gene editing (132). There is also an uncertainty about the potentially harmful effects on the offspring, especially when off-target effects occur in the germline (133). Another potential issue is that genome editing by CRISPR–Cas9 induces a p53-mediated immune response and cell cycle arrest (134). These and other concerns with regard to gene editing were even more ignited after the birth of the first gene-edited babies in China, when He Jiankui acted against official regulations (released by China’s health and science ministries in 2003) as announced at the Second International Summit on Human Genome Editing in Hong Kong on 29 November 2018 (135). Gene editing has thus been the subject of many ethical debates and concerns worldwide. Unfortunately, rules and legislation about the use and implementation of this technique are lagging behind.
These downsides of the available gene editing techniques stress the need for alternative therapeutic approaches. Sustained correction of aberrant expression levels of disease-associated genes as well as reversal of the downstream effects of a genetic mutation can be achieved through Epigenetic Editing. Extensive elaboration of the concept as well as applications of Epigenetic Editing are discussed throughout this thesis. Through Epigenetic Editing a natural, endogenous expression control is exerted by epigenetic rewriting of the gene’s own promoters/enhancers, resembling more physiological conditions in normal cells (136). Further, multiple isoforms of a gene can be targeted, which could be essential in achieving the desired functional outcome (137). Even though Epigenetic Editing suffers from the same downside with off-target effects as genetic editing, its reversible nature allows for an easier correction or reversal of potential side-effects. Unwanted edited epigenetic marks can be removed or replaced again in a targeted and gene-specific manner, without long-lasting effects on the gene or DNA sequence. Despite this reversible nature, edited epigenetic marks can be inherited by the next cell generation, and stably maintained throughout cell
divisions (71, 75, 111), resulting in durable and long-lasting modification of gene expression. This is different from epigenetic marks being inherited by the offspring, which is only possible through alterations within the germ cells (the egg and sperm). This is highly controversial and is not, nor should it be, allowed in humans. Being inherited by the next cell generation however, is important for clinical purposes where the ideal therapy would be to administer Epigenetic Editing constructs in a one-and-done approach, where patients are treated once, after which the modified epigenetic marks are remembered and maintained on the target gene. In this way, the final result of Epigenetic Editing on gene expression can be similar to gene editing approaches with regard to permanent reprogramming of gene expression, but now without changing the DNA sequence.
The ability to permanently reprogram gene expression is especially apparent during mammalian development. Even though most cells within an organism contain the same DNA, there are many different cell types, making up the various tissues and organs. Epigenetic modifications underlie cell identity by switching cell-type specific genes on or off during cell divisions (e.g. proliferating liver cells, remain liver cells) (138). Because of this intrinsic mechanism of maintenance of epigenetic marks, also edited epigenetic marks can remain stable on the DNA or histone tails, even after removal of the Epigenetic Editing tool. Furthermore, the extend of the rewritten epigenetic marks can spread along the target gene (139), as endogenous epigenetic enzymes are recruited to the target side (140, 141), also contributing to the maintenance of the rewritten epigenetic environment. Taken these considerations into account, even though it has not yet evolved into a clinical translation such as gene editing, Epigenetic Editing is ethically considered less invasive (safer) as no genetic changes are introduced. Reports on preclinical therapeutic effects upon viral delivery of epigenetic editors together with an improved understanding of biology, are facilitating in vivo Epigenetic Editing and transcriptional modulation (79). For example, Fragile X syndrome (FXS), the most common genetic form of intellectual disability in males and often associated with social anxiety and autism, is caused by silencing of the FMR1 gene by DNA hypermethylation. Removing of these methyl groups by lentiviral delivery of dCas9-Tet1 (targeted demethylation) restores the expression of FMR1 in multiple FXS patient derived induced pluripotent stem cell (iPSC) lines. After maturation of these stem cells into neurons (by well-established differentiation protocols) and transplanting them into the brains of new-born
mice, the matured neurons continued to express FMR1 three months later (142). The same approach could have promise for treating Rett syndrome, which generally is lethal in boys and related with autism in girls, and results from a loss-of-function mutation in one copy of the X chromosome-linked gene MECP2. Re-expressing specific regions in the normally silenced X chromosome could turn on the intact version of MECP2, without affecting other X chromosome genes, to treat Rett syndrome symptoms (143). Another in vivo example of preclinical effects using Epigenetic Editing is provided by Zeisberg et al., who show that re-expression of hypermethylated genes Rasal1 and Klotho, using lentiviral delivered dCas9-Tet3, attenuates disease progression in a mouse model of kidney fibrosis (144). Hypermethylation of these genes has also been associated with fibrosis in heart (145) and liver (146) as well as progression of various forms of cancer (147), demonstrating that CRISPR-dCas9-based gene-specific DNA demethylation has a broad application spectrum and may be useful to combat these other diseases as well. In neurodegenerative disorders like e.g. Alzheimer’s disease, targeted upregulation of Dlg4 has been shown to lead to functional differences like improved learning in aged and Alzheimer's disease mice (136). Injection of herpes simplex virus (HSV) particles containing ZF-VP64 constructs targeted to the Dlg4 gene into the hippocampus of these mice, improved their performance in learning-related tasks and rescued memory deficits. These, and other (45, 63, 71-73, 92, 93, 148-153), examples show the potential of using Epigenetic Editing and targeted transcriptional modulation as a therapy to treat human diseases (Table 1).
Table 1: Noncomprehensive summary showing the therapeutic potential of using Epigenetic Editing in vivo to treat
human diseases
Human Disease Purpose Platform + Targeting
ED Delivery method Result Animal Reference
Breast Cancer Targeted DNA methylation of SOX2 ZF-DNMT3A
Xenograft transplantation of
tumour cells that stably express the
constructs
Long-lasting oncogenic repression and DNA
methylation Adult Mice Stolzenburg et al. 2015 (92) Several human diseases
and biological processes
Develop strategy for targeted DNA
demethylation dCas9-TET1 Lentiviral
Activation of a methylation reporter by demethylation of
its promoter
Adult
Mice 2016 (148) Liu et al.
Cancer and repression ofTranscriptional activation cancer-related genes
dCas9 dCas9-VP64
Injection of cells that stably
express the constructs
Down-regulation of Trp53: accelerating disease onset, reducing survival, resistance to
treatment + Increased expression of
MGMT: resistance to
treatment and shorter survival Adult Mice
Braun et al. 2016. (149)
Alzheimer’s disease Transcriptional activation of Dlg4 ZF-VP64 Herpes Simplex Viral
Upregulation of Dlg4 and improved performance in learning-and memory-related
tasks
Adult
Mice Bustos et al. 2017 (136) Several human diseases,
including cancer
Develop strategy for locus-specific, rapid and efficient
DNA methylation dCas9- M.SssI-Q147L Zygote micro-injection of plasmids
Targeted DNA methylation at specific CpGs of the imprinted
locus Igf2/H19
Mice
embryo’s Lei et al. 2017 (4) Several human diseases
including type I diabetes, acute kidney
injury, and muscular dystrophy
Transcriptional activation
of endogenous genes MS2/P65/HSF1 Adeno-Associated Viral
Rescue levels of gene expression (e.g. Klotho) +
Compensate for genetic defects (Utrophin) + Alter cell
fates (Pdx1)
Adult
Mice Liao et al. 2017 (62) Fragile X syndrome Re-expression of the silenced FMR1 gene dCas9-Tet1 Lentiviral Targeted demethylation and re-expression of FMR1 Adult Mice Liu et al. 2018
(142) Kidney fibrosis hypermethylated genes Re-expression of
Rasal1 and Klotho dCas9-Tet3 Lentiviral
Gene specific demethylation and re-expression + attenuation of fibrosis
Adult
Mice Xu et al. 2018 (144) Retinitis Pigmentosa Targeted repression of Nrl dCas9-KRAB Adeno-Associated Viral the retina + reprogramming of In situ Nrl gene repression in
rods into cone-like cells
Adult
Mice al. 2018 (72) Moreno et
Liver injury Transcriptional activation of hepatocyte-
specific genes in the liver dCas9-VP64
Adeno-Associated Viral in dCas9 Mice
Activation of endogenous gene expression, development of
hepatocellular carcinomas when oncogenes were
activated Adult Mice Wangensteen et al. 2018 (150) Investigation of gene function in the mammalian brain Targeted repression of
genes in neurons dCas9-KRAB
Lentiviral, stereotactic
injections
Inactivation of genes fundamental for neurotransmitter release with
high efficiency
Adult
Mice al. 2018 (152) Zheng et
Obesity Transcriptional activation of Sim1 and Mc4r dCas9-VP64 Adeno-Associated Viral
Increased expression of Sim1 and Mc4r + Reversal of the
obesity phenotype in haploinsufficient mice
Adult
Mice al. 2019 (93) Matharu et
Neuropsychiatric diseases, such as addiction, depression, schizophrenia, and Alzheimer’s disease Transcriptional activation of targeted genes in neurons dCas9-VP64 Lentiviral, stereotactic injections
Specific and large-scale control of gene expression profiles within the central nervous
system
Adult
Mice Savell et al. 2019 (151) Breast Cancer Transcriptional activation of tumour suppressor
genes dCas9-VP64 Dendritic Polymers
Reactivation of MASPIN and
CCN6
+ cancer growth inhibition
Adult
Concluding Remarks
In order to propel Epigenetic Editing towards clinically addressing human pathologies, the challenges discussed in this thesis should be taken into account. Overcoming critical challenges such as chromatin context-dependency, delivery and specificity will be essential in the coming years and at the same time public debate about the applications and ethical considerations should be encouraged. Science is not finished until it is communicated, and only through outreach can science truly make an impact. As researchers, we all have the obligation and responsibility to act in an ethically responsible way, to educate the future generation and to engage with society. Bridging the gaps in research, communication and legislation will ultimately decide the success of “the curable genome” using Epigenetic Editing in human diseases.
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