REGULATION

Cross-Reference to Related Applications

This Patent Appl ication claims priority from Italian Patent Application No . 102022000006296 filed on March 30 , 2022 , the entire disclosure of which is incorporated herein by reference .

Technical Field of the Invention

The present invention relates to a gene regulating system comprising : 1 ) a functional nucleic acid molecule comprising : a first portion comprising at least one sequence coding for a dCAS 12 f protein lacking nuclease activity and maintaining the ability to anchor to a target DNA sequence , and at least one second portion comprising a sequence coding for an epigenetic enzyme or enzymatically active portion thereof ; and at least one promoter upstream of the first portion; and 2 ) a guide RNA or DNA encoding therefor, which is capable of recogni zing the target DNA sequence and directing said dCAS 12 f protein thereto , wherein the functional nucleic acid molecule and the DNA encoding for the guide RNA are included in the same nucleic acid molecule .

Prior Art

Between 3 . 5% and 5. 9% of the world population is af fected by one of circa 6000 known rare diseases . Gene mutations may alter the expression of genes and indirectly of multiple genes . However, additional biochemical mechanisms , known as epigenetic writers and erasers , exert fine control of virtually all genes ' expression independently of their DNA sequences. Epigenetic modifications include delicate physiological regulatory mechanisms through different developmental phases.

Methylation at 5-cytosine (5mC) is a natural DNA biochemical modification. The 5mC methylation is a dynamic and reversible process mainly mediated in humans by DNA methyltransferase (DNMT-1, DNMT-3A, and DNMT-3B) . On the contrary, the demethylation is primed by Ten-eleven translocation (TET) that oxidates the 5mC to 5- hydroxymethylated C (5-hmC) . The 5-hmC represents the starting signal for a further cascade of reactions that lead to the unmethylated cytosine. DNA Methylation regulates the accessibility of chromatin, the binding of specific transcription factors to the gene promoter, and the assembling of the transcription machinery. For these reasons, aberrant methylation in many gene promoters leads to a wide range of abnormal organism responses, from tumours to psychiatric symptoms, insomnia or hypersomnia, and immune system suppression. Developing efficient sequence-specific methylation and demethylation technologies may have significant clinical benefits. To date, only a few epigenetic drugs have been approved for human treatment in neurological diseases and cancers (Mann et al. 2007, Mikaelsson et al., 2011) . However, current methods of manipulating DNA methylation are primarily based on global inhibition of DNA methyltransferases via small molecule compounds such as hypomethylating agents, including Azacitidine or Decitabine. These molecules behave as a false substrate that replaces the DNA cytosine with a similar nucleoside (e.g., Cytidine) that cannot be methylated. During the DNA replication, the progressive substitution of cytosines leads to broad epigenetic changes and activation of endogenous retroviruses. As a consequence of this global constitutive methylation, throughout the whole organism, side effects related to off-target organs or cell types are recurrent.

In recent years we witnessed a fast revolution in geneediting technologies such as the combination of clustered regularly interspaced palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins. CRISPR technology is based on DNA breaks and homology-directed repair mechanisms, which can be programmed by noncoding single guide RNAs (sgRNAs) to guide specific nuclease (Cas9) to induce site-specific DNA cleavage in virtually any cellular system and organism. Despite the enormous potential of DNA editing in gene therapy, single-strand nicks bring significant potential cellular toxicity, making these approaches less suitable for therapeutic strategies. Moreover, CRISPR application in human disease treatments presents a number of limitations that still prevent its application in most human gene therapy .

It has been shown that by combining catalytically inactive Cas9 (dCas9) to parts of human DNMT (Stepper et al. 2017) and bacterial methyltransferases (Lei et al. 2017) or human TET1 catalytic domains, the system can be used to drive the epigenetic writers on a specific sequence of the DNA.

These approaches have just been developed and still have been tested limitedly.

There is a need to further develop this approach in order to obtain robust and reliable systems for controlling the expression of genes by modulating their epigenetic landscape without introducing changes to the underlying DNA sequences in vivo and in particular, in human patients .

Summary of the Invention

It is an obj ect of the present invention to provide a gene regulating system that allows to precisely and timely control expression of target genes by epigenetic modi fications without introducing changes of the underlying DNA sequences . This invention is instrumental in preventing and correcting human disorders as well as developing ad hoc treatments for common symptoms across diseases , overall developing new classes of precise gene-based treatments ( e . g . , precise epi-gene therapy) .

This obj ect is achieved by means of the gene regulating system as defined in claim 1 .

Other obj ects of the present invention are to provide medical uses of the gene regulating system as defined in claims 5 to 9 .

Definitions

By " functional nucleic acid molecule" there is generally intended that the nucleic acid molecule is capable of inducing methylation or demethylation of a target DNA of interest . In particular, this activates or inactivates gene expression by modi fying the epigenetic elements that regulate the transcriptional machinery of the target gene .

The "CRISPR" is a microbial immune system involved in defense against invading exogenous DNA material . The CRISPR loci in microbial hosts contain a combination of CRISPR- associated (Cas) genes as well as noncoding RNA elements capable of programming the specificity of the CRISPR- mediated nucleic acid cleavage. Cas9 forms a complex with the 3' end of the single guide RNA ("sgRNA") , and the protein-RNA pair recognizes its genomic target by complementary base pairing between the 5' end of the sgRNA sequence and a predefined 20 bp DNA sequence, known as the protospacer. By simply exchanging the 20 bp recognition sequence of the expressed chimeric sgRNA, the Cas9 nuclease can be directed to new genomic targets. CRISPR spacers are used to recognize and silence exogenous genetic elements in a manner analogous to RNAi in eukaryotic organisms. An engineered form of the effector system of Streptococcuspyogenes was shown to function in human cells for genome engineering. The Cas9 protein can be directed to genomic target sites by a synthetically reconstituted "guide RNA" ("gRNA") . Target recognition occurs upon detection of complementarity between a "protospacer" sequence in the target DNA and the remaining spacer sequence in the gRNA. Cas9 mediates cleavage of target DNA if a correct protospacer adjacent motif (PAM) is also present at the 3' end of the protospacer. For protospacer targeting, the sequence must be immediately followed by the protospacer-ad acent motif (PAM) , a short sequence recognized by the Cas9 nuclease that is required for DNA cleavage. Different Type IT systems have differing PAM requirements. The S. pyogenes CRISPR system may have the PAM sequence for this Cas9 (SpCas9) as 5 ' -NRG- 3' , where R is either A or G, and characterized the specificity of this system in human cells. A unique capability of the CRISPR/Cas9 system is the straightforward ability to simultaneously target multiple distinct genomic loci by co-expressing a single Cas9 protein with two or more s gRNAs .

"CRISPR-Casl2f " , is a family of exceptionally compact RNA-guided nucleases from uncultivated archaea. Originally identified as a single-stranded DNA (ssDNA) cutter, the wildtype Casl2f (also known as Casl4) system was recently discovered to possess PAM sequences for double-stranded DNA cleavage in vitro. The Casl2f system has no detectable activity in mammalian cells but RNA engineering and protein engineering can be applied to this isoform to generate a compact, efficient, and specific system for mammalian genome engineering (which is also referred to as CasMINI) . This provides a new method to engineer compact and efficient CRISPR-Cas effectors that can be useful for broad genome engineering applications, including gene regulation, gene editing, base editing, epigenome editing, and chromatin imaging. The Casl2f can be mutated to generate amino acid substitutions D326A and D510A and obtain the nuclease- inactivated dCasl2f. Casl2f and its derivate dCasl2f possess a TTTR PAM. dCasl2f can be ligated to epigenetic writers to modify the epigenetic regulation of specific target.

"MSssI" or "MQ1" refers to the DNA methylase of Spiroplasma sp . strain MQ1, which displays the same sequence specificity as that of the known mammalian DNA methylases. The sequence specificity of this methylase is characterized by methylating exclusively CpG sequences, making it a convenient tool for studying DNA methylation in eukaryotes. MSssI, a CpG methylase is the first discovered prokaryotic DNA methylase able, firstly to recognize CpG dinucleotide sequence and it shares this recognition sequence with the eukaryotic DNA methylase. Spiroplasma is a plant parasite and it is possible that this bacteria inherited the gene from its eukaryotic host.

"Ten-eleven translocation methylcytosine dioxygenase 1 (TET1)" is a member of the TET family of enzymes, and in humans, it is encoded by the TET1 gene. The full structure of TET1 includes a double-stranded p-helix (DSBH) domain, a cysteine-rich domain, and a CXXC domain. The dioxygenase activity of DSBH is dependent on Fe2+ and 2-oxoglutarate (2- OG) . Double-stranded /? -helix is the active catalytic subunit, which oxidizes the methyl group attached to the 5' position of C. The function of the cysteine-rich domain is to stabilize the TET-DNA interaction. Its CXXC domain recognizes and binds to unmethylated CpG sites. TET1 catalyses the hydroxylation of DNA methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) , and it can further oxidize 5hmC into 5-formylcytosine (5fC) and 5-carboxycytosine (5caC) . However, 5fC and 5caC are mostly unstable, as they can be rapidly excised by thymine DNA glycosylase (TDG) in an active de-methylation state. Moreover, TET1 directly interacts with transcription factors and histone modification enzymes to regulate gene expression.

Brief Description of the Drawings

Figure 1 shows a schematic representation of the generation of a vector (miniOFF-1) according to one embodiment of the present invention; Figure 2 shows a schematic representation of the generation of a vector ( aavOFF- 1 ) according to another embodiment of the present invention;

Figure 3 shows a schematic representation of the generation of a vector (miniON) according to another embodiment of the present invention;

Figure 4 shows a schematic representation of the generation of a vector ( aavON- 1 ) according to another embodiment of the present invention;

Figure 5 shows a schematic representation of the strategy to generate AAV containing the gene regulating system according to an embodiment of the present invention;

Figure 6 shows a schematic representation of the promoter sequence regulating the expression of the human gene MGMT and the consensus sequence for the guide included in the vector named TUSER+sgMGMT of Example 6 .

Figure 7 shows a schematic representation of the promoter sequence regulating the expression of the human gene PARP1 and the consensus sequence for the guide included in the vector named TUSER+sgPARPl of Example 6 .

Figures 8 to 11 show graphs of the methylation of the PARP1 and MGMT promoters and of the expression of the PARP1 and MGMT mRNAs .

Figure 12 shows a graph of cell viability after TUSER and temozolomide treatment .

Figure 13 shows a schematic representation of the promoter sequence regulating the expression of the human gene HCRT and the consensus sequence for the guide included in the vector named TUSER+sgHCRT of Example 7 . Figure 14 shows the endogenous expression of the cell lines used in the example 7 .

Figure 15 shows the methylation of the HCRT promoter in the Hek293T cell treated with TUSER+sgHCRT compared to the cell treated with the control vector of the example 7 .

Figure 16 shows the mRNA expression of the human HCRT gene in the Hek293T cell treated with TUSER+sgHCRT compared to the cell treated with the control vector of the example 7 .

Figure 17 shows the expression level of the TET1CD mRNA encoded in the construct aavON- 1 compared to the endogenous level of the TET in the HEK293T cells .

Figure 18 show the expression level of the dCas l2 f encoded in the aavON- 1 construct compared the public available miniCAS construct .

Detailed Description of the Invention

A gene regulating system according to the present invention comprises :

- a functional nucleic acid molecule comprising :

- a first portion comprising at least one sequence coding for a dCAS 12 f protein lacking nuclease activity and maintaining the ability to anchor to a target DNA sequence , and at least one second portion comprising a sequence coding for an epigenetic enzyme or enzymatically active portion thereof ; and

- at least one promoter upstream of the first portion; and

- a guide RNA or DNA encoding therefor, which is capable of recognizing the target DNA sequence and directing said dCAS12f protein thereto, wherein the functional nucleic acid molecule and the DNA encoding for the guide RNA are included in the same nucleic acid molecule

Preferably, dCAS12f has sequence SEQ ID NO: 7. The CAS protein is engineered to a minimal DNA sequence that can still produce a protein lacking nucleases activity and be used as DNA anchoring to target specific DNA sequences, with the epigenetic enzyme, which is preferably Spiroplasma methyltransferase MQ1 (more preferably SEQ ID NO: 8) or the catalytic domain of the human Tet Methylcytosine Dioxygenase 1 (TET1CD) (more preferably SEQ ID NO: 9) . The reduced (minimal) size of constructs makes this programmable epigenome editing technology suitable for delivery with an adeno-associated virus (AAV) . In addition, the gene regulating system may contain regulatory elements suitable for the specific tissue or cell expression in the host organisms .

In a preferred embodiment, the first portion is a fusion protein between dCasl2f, and two different epigenetic writers such as the sssIM de novo methyltransferase from MQ1 Spiroplasma or the catalytic domain of the human Tet Methylcytosine Dioxygenase 1 (TET1CD) . The MQ1 methyltransferase catalyzes the addition of a methyl group (CH3) to the cytosine of the cytosine-guanidine dinucleotide (CpG) in the DNA sequence. By contrast, the TET1CD protein catalyzes the modification of the methyl-cytosine in the DNA to a hydroxyl-methyl-cytosine leading to the removal of the methyl group from the cytosine residue. The methylation of a gene promoter induces robust gene silencing, whereas the de-methylation of such sequence activates the gene expression. The recruitment of MQ1 or TET1CD on the gene promoter may positively and negatively modulate the gene expression, respectively.

The at least one promoter upstream of the first portion is preferably a CMV or PGK promoter. More preferably the CMV promoter has sequence SEQ ID NO:1 and the PGK promoter has sequence SEQ ID NO: 2.

The product of the above said functional nucleic acid molecule is directed to the specific DNA target by administering specific guide RNA.

The guide RNA is a specific RNA sequence that recognizes the target DNA region of interest and directs the Gas nuclease there for editing. The gRNA is made up of two parts: crispr RNA (crRNA) , a 17-20 nucleotide sequence complementary to the target DNA, and a tracr RNA, which serves as a binding scaffold for the Gas nuclease. The Gas nuclease binds to its target sequence only in the presence of a specific sequence, called protospacer adjacent motif (PAM) , on the non-targeted DNA strand flanking the consensus sequence of the guide. In the case of dCAS9, the 17-20 nucleotides targeted sequence must be flanked by three nucleotides 5 ^Z -NGG-3 (where N consists in any nucleotide) . Overall the guide must be composed of the targeting sequence (herein referred as guide) , the PAM motif, and the relative scaffold sequence to anchor the guide to the respective Gas. The overall length of the guide ranges from 60 to 80 bases and the relative cDNA sequence can be cloned in an RNA- expressing vector under the control of RNA polymerase I I I promoters such as U6 .

The gene regulating system according to the invention therefore comprises the above disclosed functional nucleic acid and a guide RNA or DNA encoding therefor, which is capable of recogni zing the target DNA sequence and directing said CAS protein thereto .

This system is referred to hereinafter also as TUSER ( targeting unit for speci fic epigenetic remodeling) and has the ability to enhance or repress speci fic gene transcription in cells without changes of the underlying DNA sequences . TUSER can be used to target a large set of genes within the mammalian genome .

Modules of this system exploit Cas ability to bind a DNA sequence after the administration of a target-speci fic RNA guide . Opposite epigenetic writers substitute dCas 9 cleaving activity characteri zing the core elements of this system : Meth-OFF and Meth-ON cassettes .

As mentioned, the reduced dimension of the TUSER constructs gives the possibility to include speci fic guideexpressing cassettes in the same AAV . In other words , the DNA encoding for a guide RNA, which is capable of recogni zing the target DNA sequence and directing said CAS protein thereto , and the functional nucleic acid molecule are preferably included in the same nucleic acid molecule , preferably an adeno-associated virus (AAV) vector .

Preferably, the DNA encoding for the guide RNA, expressed by a speci fic U6 promoter that recruits the RNA polymerase class I I I , is cloned downstream the epigenetic writer cassette in the opposite direction with respect to the coding sequence .

This strategy induces epigenetic remodeling mediated by the administration of a single AAV containing both the epigenetic writer and the guide for a particular genetic region to treat a range of diseases .

The constructs according to the present invention are preferably designed to be suitable for adeno-associated virus packaging . During the past decade , in vivo administration of viral gene transfer vectors for the treatment of numerous human diseases has been brought from bench to bedside in the form of clinical trials , mostly aimed at establishing the protocol ' s safety . In preclinical studies in animal models of human disease , adeno-as sociated viral (AAV) vectors have emerged as a favored gene trans fer system for this approach . These vectors are derived from a replication-deficient , non-pathogenic parvovirus with a single-stranded DNA genome . Ef ficient gene trans fer to numerous target cells and tissues has been described . AAV is particularly ef ficient in the transduction of non-dividing cells , and the vector genome persists predominantly in episomal forms . Substantial correction, and in some instances complete cure , of genetic disease , has been obtained in animal models of hemophilia, lysosomal storage disorders , retinal diseases , disorders of the central nervous system, and other diseases , including brain tumors .

In particular, glioblastoma is one of the most malignant primary brain tumours with an aggressive phenotype and a poor very short survival rate outcome caused by the inef fectiveness of current treatments . Temozolomide ( TMZ ) represents the current standard of treatment . By al kylating guanine residues , TMZ induces futile DNA mismatch repair that introduces single-stranded breaks resulting in replication fork arrest and cell death . Two main DNA repairing programs in the cells cause the dominant mechanism of TMZ resistance . The DNA repair enzyme 06-methylguanine- DNA methyltrans ferase (MGMT ) removes alkylated guanine bases to counter the lethal ef fects of the drug . Similarly, poly (ADP-ribose ) polymerase 1 ( PARP1 ) expression enhances the base scissor repair system repairing DNA breaks provided by TZM ( Fig) . Hence , cancers in which MGMT or PARP1 expression is suppressed by methylation tend to be highly responsive to TMZ , with an increase in median survival of approximately six and one-hal f months compared to less than one month for cancer expressing these proteins . However, MGMT promoter hypermethylation occurs in less than 50% of primary GBM patients , whereas PARP1 is normally expressed in the cells .

The gene regulating system according to the present invention has been trans fected into primary GBM prior to TMZ treatment cells to increase MGMT or PARP1 promoter methylation and hij ack GBM resistance .

The gene regulating system according to the present invention can therefore be used as a medicament .

In particular, the gene regulating system can induce upregulation or downregulation of the expression of a gene in a human individual in which said gene is respectively pathologically underexpressed or overexpressed .

Preferably, the gene regulating system according to the present invention is used in treating cancer . The cancer is preferably glioblastoma . The gene regulating system according to the present invention can also be applied to reinforce speci fic treatments . Preferably, it can be used as a strategy to prevent chemotherapy resistance , such as glioblastoma multi forme ( GBM) treatment with temozolomide . The gene regulating system according to the present invention can also be applied to gene haploinsuf f iciency or overexpression cases .

The technology according to the present invention was validated by designing experimental applications that have the potential to impact various physiological domains such as cancer biology .

Examples

The following constructs were generated by conventional techniques .

Example 1 - mini-OFF-1

Casl2f small CRISPR-associated effector proteins belong to the type V-F subtype of the Cas family. The sequence encoding for a nuclease-deactivated Casl2f (dCasl2f) is commercially available as a bacterial plasmid (Addgene inc., plasmid #176269) where a PGK promoter expresses the coding sequence. With reference to Figure 1, the binding site for the endonuclease Nhel present in the vector was inactivated by site direct mutagenesis, downstream the coding sequence, and introduced the binding site for this endonuclease at 3' position of the miniCAS coding sequence. The vector (Addgene inc, plasmid #89793) was mutated to introduce the binding sequence for the endonucleases Spel at the 5' end of the MQ1 sequence, and the binding sequence for the endonuclease EagI the 3' end of the sequence.

The PGK-dCasl2f was treated with Nhel and EagI, whereas the mutated MQ1 plasmids were treated with Spel and EagI . The final vector was obtained by ligating the Nhel-PGK- dCasl2f-EagI fragment with the Spel-MQl-Eagl fragment to obtain the final construct miniOFF-1.

Example 2 - aavOFF-1

With reference to Figure 2, the sequence of miniOFF-1 was amplified by PGR using a set of two primers, presenting the binding sequence for Nhel and BamHI, respectively. The amplified was used to include the miniOFF-1 in the pAAV-MCS2 vector (Addgene Plasmid #46954) . Both the pAAV-MCS2 vector and the amplified were treated with Nhel and BamHI endonucleases and the NheI-pAAV-MCS2-BamHI fragment was ligated to the Nhel-miniOFF-l-BamHI fragment to obtain the aavOFF-1. Finally, a synthetic cassette containing the U6 promoter followed by the guide sequence and a small terminator sequence was cloned downstream the transcriptional terminator WPRE in the opposite direction respecting the dCasl2f-MQl coding sequence.

Example 3 - miniON-1

With reference to Figure 3, as described for the construct miniOFF-1, the binding site for the endonuclease Nhel downstream the coding sequence was inactivated by mutation and the binding site for this endonuclease introduced at 3' position of the miniCAS coding sequence. The catalytic domain of the TET1 gene was cloned by PCR by two pairs of primer containing the Spel and the EagI binding sites .

The PGK-dCasl2f was treated with Nhel and EagI, whereas the amplified TET1CD sequence was treated with Spel and EagI. The final vector was obtained by ligating the Nhel-PGK- dCasl2f-EagI fragment with the SpeI-TS-ON-3-EagI fragment to obtain the final construct miniON-1 (Fig. 9G) .

Example 4 - aavON-1

With reference to Figure 4, the sequence of miniON-1 was amplified by PCR using a set of two primers, presenting the binding sequence for Nhel and for BamHI, respectively. The amplified was used to include the miniON-1 in the pAAV- MCS2 vector. Both the pAAV-MCS2 vector and the amplified were treated with Nhel and BamHI endonucleases, and the Nhel- pAAV-MCS2-BamHI fragment was ligated to the NheI-miniON-1- BamHI fragment to finally obtain the aavON-1. Finally, a synthetic cassette containing the U6 promoter followed by the guide sequence and a small terminator sequence was cloned downstream the transcriptional terminator WPRE in the opposite direction respecting the dCas l2 f-TETlCD coding sequence .

Example 5 - applications for AAV-OFF and AAV-ON

The mini-OFF and mini-ON vectors are instrumental to the creation of the Adeno Associated Virus (AAV) delivery strategy . AAVs are one of the most actively investigated gene therapy vehicles . They are non-enveloped viruses that can be engineered to deliver DNA to target cells and have attracted signi ficant attention in the field, especially in clinical-stage experimental therapeutic strategies . The ability to generate recombinant AAV particles lacking any viral genes and containing DNA sequences of interest for various therapeutic applications has thus far proven to be one of the safest strategies for gene therapies .

The small ( 4 . 8 kb ) ssDNA AAV genome consists of two open reading frames , Rep and Cap, flanked by two 145 base inverted terminal repeats ( ITRs ) . These ITRs base pair to allow for the synthesis of the complementary DNA strand . Rep and Cap are translated to produce multiple distinct proteins (Rep78 , Rep68 , Rep52 , Rep40 , required for the AAV li fe cycle and VP1 , VP2 , VP3 for the capsid proteins ) . When constructing an AAV trans fer plasmid, the transgene is placed between the two ITRs , and Rep and Cap are supplied in trans .

In addition to Rep and Cap, AAV requires a helper plasmid containing genes from adenovirus . These genes (E4 , E2a, and VA) mediate AAV replication . The trans fer plasmid, Rep/Cap, and the helper plasmid are trans fected into producing cells (e.g., HEK293 cell line) , which contain the adenovirus gene E1+, to produce infectious AAV particles. Rep/Cap and the adenovirus helper genes may also be combined into a single plasmid; the separation of Rep and Cap shown in the figure on the right facilitates the viral pseudotyping discussed below.

In particular, the mini-OFF or the mini-ON coding TUSER sequence and the guide of the Casl2f to the specific target gene can be cloned between the two ITRs and packaged into the final AAV particles. The AAV-OFF and AAV-ON vectors were produced by cloning the mini-OFF and mini-ON cassette into a pAAV-MCS2 vector. This vector encodes the genes necessary to produce a serotype 2 AAV (AW2) . This serotype is suitable for transducing the genetic content specifically into the central nervous system, kidney, or retinal photoreceptor cells. Moreover, the AAV production technique can be instrumental in producing any commercial AAV serotype by replacing the pAAV-MCS2 with the respective serotype vector.

Moreover, the coding sequence of aavOFF and aavON vectors contain the dCasl2f fused to the respective MQ1 or TET1CD coding sequence. In the 3' flank of this sequence, a WPRE sequence terminates the epigenetic writer's transcription and stabilizes the produced mRNA.

In an AAV-mediated strategy for treating the human disease, a cassette expressing the gRNA for a specific target was included. Such cassettes can be composed of a U6 promoter, the coding sequence of the guide and a small transcriptional terminator sequence.

The U6 promoter recruits the RNA polymerase III to transcribe the gRNA sequence . Such a designed construct can be used to create an AAV expressing both the epigenetic writer and the target-speci fic guide in the same delivery vector .

Example 6 - TMZ treatment

Gliomas are the most common primary brain tumours in adults . Despite the progress made in the molecular aspects of malignant gliomas , the prognosis of brain tumours continues to be dismal . Glioblastoma multi form ( GBM) is a highly aggressive brain tumour with a median patient survival of 14 . 6 months . Chemotherapy has been used for decades to treat GBM, and the oral methylator temozolomide ( TMZ ) is currently the most widely used chemotherapeutic agent . TMZ achieves cytotoxicity mostly by methylating the 06 position of guanine ( 0me 6G) . The 06-methylguanine DNA methyltrans ferase (MGMT ) can reverse alkylation at the 06 position of guanine and neutrali ze the cytotoxic ef fects of alkylating agents . MGMT expression in brain tumors represents a key mechanism of resistance toward alkylating agent therapy . Moreover, TMZ induces not only 0me 6G but also large numbers of N3-methyladenine and N7 -methylguanine adducts that are rapidly processed by the base excision repair (BER) DNA repair pathway . N3-methyladenine and N7- methylguanine lesions activate the nuclear enzyme poly-ADP- ribose-polymerase 1 ( PARP1 ) , which synthesi ze poly-ADP- ribose chains and facilitate the recruitment of XRCC1 , pol- beta, and DNA ligase to repair the DNA strand break sites .

In this example the transcription of MGMT and PARP1 are inhibited selectively, inducing the methylation of the promoter region of such genes with the construct aavOFF-1.

We included in the example three different glioblastoma cell line, U87, U118 and T98G

Guide Design sgRNA sequences for each gene were designed to target human MGMT (5' -CGCTGCCGGAGGACCAGGGCCGG-3' - SEQ ID NO: 11) and PARP1 (5' -CCAAAGAGCTACTAGCTCAGCC-3' - SEQ ID NO: 10) promoter sequences. MGMT sgRNA consists of a guide targeting the consensus sequence at 248 bases upstream of the MGMT TSS (FIGURE 6, sgRNA-1 - dark arrow) , whereas PARP1 is designed to target the consensus sequence located 338 bp upstream the TSS of the PARP1 gene (FIGURE 7, sgRNA-1 - dark arrow) . Each guide was included in the vector coding for the epigenetic writer named aavOFF-1 (Figure 2) , here referred as TUSER+sgMGMT and TUSER+sgPARPl , and used to treat the glioblastoma cell lines. The control vectors were produced by delating the relative sgRNA guide of the plasmids.

Cell lines and transfection

U118 cell line, procured from the American Tissue Collection Center (ATCC, Manassas VA) , was cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS and 1% penicillin/streptomycin and maintained at 37°C and 5% C02. Transfections were performed in 12-well plates using 1000 ng of the respective TUSER+sgMGMT and TUSER+sgPARPl expression vector mixed with Lipof ectamine 2000 (Life Technologies, cat. # 11668019) as per manufacturer's instruction.

Methylation assay

The methylation assay uses a pair of restriction enzymes, MspI and Hpall with different sensitivity to methylation to analyze DNA methylation status at a specific sequence. MspI and Hpall are isoschizomers with CCGG specificity. When the internal CpG in 5'-CCGG-3' tetranucleotide is methylated, cleavage with Hpall is blocked, but cleavage with MspI is not affected.

According to the manufacturer guidelines, genomic DNA samples were extracted from the trated cells using Trizol reagent (thermofisher) . 1 ug of the samples' genomic DNA was incubated for 1 hour at 37 °C with the two restriction enzymes separately. 25 ng of treated samples were used as templates in a qPCR analysis.

The percentage of methylation was calculated with the formula :

% of 5-mC =100 / (1 + E)Cq2-Cql

Where :

Cql is the threshold cycle of the "Undigested DNA" sample, nCq2 is the threshold cycle of the "Digested with Hpall" sample, Cq3 is the threshold cycle of "Digested with Epi MspI" sample, and E is the PGR efficiency value (%) mRNA expression by qPCR

Total RNA was extracted from treated cells cells with Trizol (Thermofisher) following the supplier's instructions, and cDNA was synthesized using 1 pg of total RNA to obtain the cDNA, with the high-capacity transcription kit following the supplier's instruction (Life Technologies) . Quantitative real-time PGR was performed with SyberGreen assays in a 7500 real-time PGR system (Life Technologies) . Changes in mRNA levels were determined as the difference in threshold cycle (2''-AACt) between the target gene (PARP1 or MGMT; primer list) and the reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) . TMZ treatment

Gliomas are the most common primary brain tumors in adults. Despite the progress made in the molecular aspects of malignant gliomas, the prognosis of brain tumors continues to be dismal. Glioblastoma multiform (GBM) is a highly aggressive brain tumor with a median patient survival of 14.6 months. Chemotherapy has been used for decades to treat GBM, and the oral administration of temozolomide (TMZ) is currently the most widely used chemotherapeutic agent. TMZ achieves cytotoxicity mostly by methylating the 06 position of guanine (0me6G) . The 06-methylguanine DNA methyltransferase (MGMT) can reverse alkylation at the 06 position of guanine and neutralize the cytotoxic effects of alkylating agents. MGMT expression in brain tumors represents a key mechanism of resistance toward alkylating agent therapy (GIT MGMT Zhi-Kun Qui) . Moreover, TMZ induces not only 0me6G but also large numbers of N3-methyladenine and N7-methylguanine adducts that are rapidly processed by the base excision repair (BER) DNA repair pathway. N3- methyladenine and N7-methylguanine lesions activate the nuclear enzyme poly-ADP-ribose-polymerase 1 (PARP1) , which synthesize poly-ADP-ribose chains and facilitate the recruitment of XRCC1, pol-beta, and DNA ligase to repair the DNA strand break sites.

In this example the transcription of MGMT and PARP1 was inhibited selectively, inducing the methylation the promoter region of such genes with the construct TS-OFF-1. Three different glioblastoma cell line, U87, U118 and T98G were included in the experiment. Results are shown in Figures 8, 9, 10 and 11. Temozolomide (MERK) was resuspended in

Dimethylsulphoxide (DMSO) to a final concentration of 50 mM. 25.000 cells per well of U87, U118 and T98G cell lines were plated in 24-well plates. The cells were treated with vehicle, lOuM, lOOuM or lOOOuM of TMZ, respectively, and cell viability was calculated to evaluate the chemotherapy toxicity .

Treatment of U87, U118, and T98G transfected cells with TZM

Transfected cells were treated with 0.5 uM of TZM, 48 after the transfection, and incubated for 48 hours before further analysis.

Cell Viability

Cell viability assay was performed with automated cell counter LunaTI (Twinx Helix inc.) .Treated cells were collected from the experimental plates and placed on the counting chip according to the manufacturer protocols. Cell counting were represented number of cell counted in each condition and compared to the sample treaded with TUSER-Mock + TMZ without the guide.

Results The U118 cells were transfected with the vectors TUSER+sgMGMT, TUSER+sgPARPl and the Mock vector encoding for the TUSER aavOFF-1 where the respective guide sequences were deleted. Both the MGMT and the PARP-1 targeting constructs were able to upregulate the methylation of the respective promoter sequence (Figures 8 and 10) , in a region between the amplification primers sequence (Figures 6 and 7) . The methylation of the promoter sequences led to a downregulation of the mRNA expression of PARP1 (Figure 9) and MGMT (Figure 11) .

Example 7: Demethylation of HCRT gene by aavON-1

Guide Design sgRNA sequences for the orexing gene was designed to target human HCRT (5' -CGCTGCCGGAGGACCAGGGCCGG-3' - SEQ ID NO: 11) promoter sequences. HCRT sgRNA consists of a guide targeting the consensus sequence at 256 bases upstream of the HCRT TSS (FIGURE 13, sgRNA-1 - dark arrow) . The guide was included in the vector coding for the epigenetic writer named aavON-1 (Figure 4) , here referred as TUSER+sgHCRT, and used to treat the HEK293T cell line. The control vectors were produced by delating the relative sgRNA guide of the plasmids .

Cell lines and transfection

HEK293T, Hela, T98G, U118 and U87 cell lines, procured from the American Tissue Collection Center (ATCC, Manassas VA) , was cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS and 1% penicillin/streptomycin and maintained at 37°C and 5% C02. The expression level of HCRT gene was measured by RT-PCR and the HEK293T cells was userd for further analysis of the TUSER+sgHCRT effect. Transfections were performed in 12-well plates using 1000 ng of the TUSER+sgHCRT expression vector mixed with Lipof ectamine 2000 (Life Technologies, cat. # 11668019) as per manufacturer's instruction.

Methylation assay

The methylation assay uses a pair of restriction enzymes, MspI and Hpall with different sensitivity to methylation to analyze DNA methylation status at a specific sequence. MspI and Hpall are isoschizomers with CCGG specificity. When the internal CpG in 5'-CCGG-3' tetranucleotide is methylated, cleavage with Hpall is blocked, but cleavage with MspI is not affected.

According to the manufacturer guidelines, genomic DNA samples were extracted from the trated cells using Trizol reagent (thermofisher) . 1 ug of the samples' genomic DNA was incubated for 1 hour at 37 °C with the two restriction enzymes separately. 25 ng of treated samples were used as templates in a qPCR analysis.

The percentage of methylation was calculated with the formula :

% of 5-mC =100 / (1 + E)Cq2-Cql

Where :

Cql is the threshold cycle of the "Undigested DNA" sample, nCq2 is the threshold cycle of the "Digested with Hpall" sample, Cq3 is the threshold cycle of "Digested with Epi MspI" sample, and E is the PGR efficiency value (%) mRNA expression by qPCR

Total RNA was extracted from treated cells cells with Trizol (Thermofisher) following the supplier's instructions, and cDNA was synthesized using 1 pg of total RNA to obtain the cDNA, with the high-capacity transcription kit following the supplier's instruction (Life Technologies) . Quantitative real-time PCR was performed with SyberGreen assays in a 7500 real-time PCR system (Life Technologies) . Changes in mRNA levels were determined as the difference in threshold cycle (2''-AACt) between the target gene (HCRT; primer list) and the reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) .

Results

The HEK293T cells were transfected with the vectors TUSER+sgHCRT and the Mock vector encoding for the TUSER aavON-1 where the respective guide sequences were deleted, the HCRT targeting constructs were able to upregulate the methylation of the promoter sequence (Figures 15) , in a region between the amplification primers sequence (Figures 13) . The methylation of the promoter sequences led to a upregulation of the mRNA expression of HCRT (Figure 16) and the expression of the TUSER construct was analysed by qPCR using forward and reverse primers amplifying the coding region of the TET1 catalytic domain (TET1CD) , included in the TUSER construct, compared to the endogenous TET1 mRNA level (Figure 17) .The expression of the TUSER construct was compared to the vector used in the CMV-SV40_NLS-CasMINI- V3. l--3xFlag-c-Myc_NLS-polyA obtained from Addgene (here named miniCAS) . This construct was produced by Xu and collegues and reported in literature (Xu, X., Chemparathy, A., Zeng, L., Kempton, H. R., Shang, S., Nakamura, M., & Qi, L. S. (2021) . Engineered miniature CRISPR-Cas system for mammalian genome regulation and editing. Molecular Cell, 81 (20) , 4333-4345.) . The analysis revealed that the expression of TUSER construct aavON-1 encoding for both the epigenetic writer and the guide was higher compared to the miniCAS in the Hek293T cell line ( Figure 18 ) .

Advantages

The present invention provides a gene regulation system that allows precise and reversible epigenetic manipulation to both in vivo and in vitro laboratory applications and provides access to gene expression manipulation for laboratories that are not prepared for complex biosafety levels of virus handling . Because of the reduced dimension of TUSER, it can be delivered by a simple adeno-associated virus (AAV) and then requiring a low biosafety level ( e . g . , BS 1 ) •

The TUSER system is customi zable , which means that by designing speci fic RNA guides that are suited to the modi fied CAS protein, it represents a novel tool to transiently modi fy DNA methylation at speci fic sites and in various experimental systems ( e . g . , inducible stem cells , primary neurons... ) . This system overcomes the di f ficulties of providing ef ficient protocols to apply CRISPR methods and, the re-combinable stop cassettes make TUSER highly speci fic in tissue expression . Overall , the presence of both methyltrans ferases and demethylases provides a flexible tool that positively or negatively regulates gene expression and its epigenetics by merely changing the chemical compound used to treat the mouse .

Declaration pursuant to Art . 170bis of the Italian Intellectual Property Code

The biological material at the basis of the invention derives from cloned synthetic molecules obtained by using information retrieved from online sequence databases .

Biological material of animal origin used in the invention comes from United Kingdom (UK) and relates to Mus musculus . Biological material of human origin used in the invention has been acquired pursuant to the applicable provisions of law . In particular, cell lines have been regularly purchased from the sources stated in the above description . In addition, any biological material containing microorganisms or genetically modi fied organisms used in the invention has been treated meeting the provisions of law relating to such modi fications .