CZ2008343A3 - Regulating gene element comprising promoter/amplifier protected from DNA methylation and gene expression suppression - Google Patents

Abstract

A promoter / enhancer regulatory gene element protected from DNA methylation and gene expression silencing, which element comprises a modified retroviral LTR, wherein one or two central IE sequences in any orientation are inserted at any LTR site, wherein the IE has the sequence set forth herein as SEQ ID NO. Preferably, at least one central IE sequence is inserted at a position between the promoter sequence and the enhancer. Such modified LTR provides sufficient and long-term expression of the integrated foreign gene vector (transgene) and its protection from silencing and sequential methylation, even in human cells.

Description

Technical field

The invention relates broadly to the field of molecular biology, virology and gene therapy, where vectors, particularly viral and retroviral vectors, are used to transfer genes to cells grown in vitro or to organisms in vivo. In particular, the invention relates to a solution to the problem of silencing gene expression and methylation of vector DNA in eukaryotic cells, particularly mammalian cells. This problem is solved by using a specific promoter / enhancer regulatory gene element that is based on the long terminal repeat sequence (LTR) of retroviruses, enriched in one or two central sequences (IE) of the CpG island. This modified LTR ensures sufficient and long-term expression of the integrated vector with the foreign gene (transgene) and its protection against silencing and gradual methylation, even in human cells. The element according to the invention has numerous applications, for example in experimental biology as a research tool, but also in clinical fields, particularly in the compensation of congenital disorders by gene therapy. It can be used in the construction of vectors for the expression of therapeutic proteins, peptides or small RNA molecules.

BACKGROUND OF THE INVENTION

For gene transfer, e.g. in gene therapy and in the preparation of transgenic animals, vectors capable of providing both the actual transfer (transduction) of foreign genes (transgenes) and their later expression are used. Viral vectors appear to be very effective (review by Osten et al., 2007; Zhang et al., 2006). Adenoviruses, adeno-associated viruses, lentiviruses or retroviruses are frequently used.

Adenoviral vectors are often designed and used for gene therapy purposes because adenovirus biology has been thoroughly investigated, viral particles are stable, and high titer adenovirus preparations can be relatively easily produced. Adenoviral vectors are capable of efficiently transducing both replicating and non-replicating cells, both in vitro and in vivo. However, the foreign DNA is present in an episomal form, so that over time it is diluted in proliferating cells and the expression of the transduced gene disappears.

Retroviruses are of particular interest in vector development, in particular because they allow for stable integration of the transgene into the genome of the host cell. Modern retro viral vectors have a large capacity for the transfer of foreign genes and thus offset the lower available titer compared to adenoviruses. They allow gene transfer to both cultured cells, tissue cultures and intact animals.

One of the advantages of retroviruses is that their life cycle alone leads to the integration of viral DNA into the host genome. When replicated, they do not have cytopathogenic effects and provide high expression in infected cells. The most commonly used retroviral vectors are derived from the replication-defective Moloney murine leukemia virus (MoMLV)

For some advantageous properties, vectors derived from avian sarcoma and leucose viruses (ASLV) are also used. The main advantage is that mammalian cells do not contain endogenous retroviruses capable of recombination with ASLV. In addition, ASLVs have an advantageous integration profile, therefore they are stable and safe in terms of possible gene damage at the site of integration or or induction of tumor growth. Of particular interest are the RCAS series vectors (Federspiel and Hughes, 1994), the replication competent vectors with splice splice derived from the Rous sarcoma virus (RSV). A significant advantage of ASLV is that they are unable to replicate in mammalian cells (Federspiel et al., 1994) because retrovirus replication requires cell cofactors for its life cycle. ASLVs cannot replicate in mammalian cells due to the lack of specific avian cofactors required for proper splicing, polyprotein cleavage, viral particle assembly, etc. (Svoboda et al., 2000). Therefore, RCAS vectors produce no infectious progeny in mammalian cells in vitro or in vivo (Federspiel et al., 1994; Barsov and Hughes, 1996). In addition, mammalian cells contain no endogenous retroviruses capable of recombination with ASLV, but a high-liter virus can be prepared using the chicken cell line DF-1, which is also free of endogenous viruses (Himly et al., 1998). Thus, ASLV-based vectors are very stable and safe for gene therapy. Another advantage of ASLV-based vectors is their integration profile, which differs from murine leukemia virus (MLV) or human immunodeficiency virus type 1 (HIV-1). Whole-genome analyzes have shown that, compared to ML V and HIV-1, ASLV exhibits the weakest preference for integration into genes and does not tend to integrate into the promoter region (Narezkina et al., 2004; Reinišová et al., 2008). Therefore, it appears to be safer than the widely used lentiviral or MLV-based vectors.

A general problem with the expression of a transferred gene into mammalian cells (in vitro and in vivo) is that the transferred gene (transgene) is generally expressed to a sufficient extent only for a relatively short period of time and then decreases in expression (transcriptional level), sometimes to completely extinguish transcription (so-called transcription silencing). The mechanisms leading to transcriptional silencing in eukaryotic cells are epigenetic, including, in particular, methylal DNA, post-transcriptional silencing (PTGS) mediated by RNAi, methylation / deacetylation of histones, and integration site effects (positional effects). These mechanisms represent a serious obstacle to the application of gene therapy. The mechanisms of DNA methylation have been extensively investigated and it is known that vertebrate DNA methylation refers to cytosine methylated at the 5 '(5mC) position in the CpG dinucleotide sequence.

CpG dinucleotides are not as abundant in the vertebrate and especially mammalian genome as would correspond to the free combination of single nucleotides. During evolution, CpG dinucleotides were eliminated by mutation and were preserved mainly in the so-called CpG islands at the beginning of some, mostly constitutively expressed genes. The CpG islands are at least 200 bp long, rich in G and C and having a CpG long nucleotide representation of at least 60% of the number we would theoretically expect with free nucleotide combinability. Usually, we find binding sites for the transcription factor Spi. Such CpG islands remain free of methylation, with the exception of CpG islands on inactivated X chromosome, imprinted loci and some aberrantly methylated CpG islands in tumor cells. The nature of the antl-methylation resistance of the CpG islands is not definitively elucidated and its knowledge is not necessary for practical application (Suzuki and Bird, 2008).

A major obstacle to the use of ASLV vectors in mammalian cells is the very efficient transcriptional silencing of the integrated provirus. It is known that retrovirus-driven reporter gene expression is not stable in vitro in the long term and gradual silencing

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The W transduced gene correlates with epigenetic changes in retroviral regulatory sequences, long terminal repeats (LTRs). The LTR sequences comprise U3, R and U5 regions whose structure and function are known. These LTR sequences contain transcription initiation and termination signals, including promoter and enhancer.

CpG methylation of DNA and / or modification of histones in nucleosomes associated with the promoter region was found in silenced proviruses in vitro. In particular, MLV and HIV-1 have been characterized in detail (Hoeben et al., 1991; Lorincz et al., 2001; Mok et al., 2007). Various anti-methylation and isolation strategies have been used to enhance the stability of MLV vectors (Rivella et al., 2000; Yannaki et al., 2002; Swindleet et al., 2004; Zhang et al., 2007).

RSV and ASLV vectors are not progressively silenced in chicken cells, however, in heterologous mammalian host cells they are effectively methylated and transcriptionally silenced (Searle et al., 1984; Hejnar et al., 1994). Protection against silencing is therefore highly needed in order to take advantage of the advantageous properties of ASLV vectors in transgenesis or gene therapy.

As mentioned above, retrovirus LTR sequences are known, e.g., the HIV-1 LTR DNA sequence has been described in U.S. Patent No. 7,232,654. The use of retroviruses for the construction of vectors suitable for the preparation of transgenic organisms has long been known. WO 91/02805. Retroviral vectors particularly suitable for expression in embryonic cells and in particular embryonic stem cells have been described, for example, in European patent EP 0 801575. Retroviral vectors suitable for gene therapy are disclosed in patent application US 2006/0153810. Modifications of LTR sequences to affect vector expression have been described in U.S. Patent No. 5,814,493. The issue of targeted silencing of gene expression in Candida albicans is discussed in International Application WO 01/48229. A specific promoter regulatory element and gene expression silencing strategies are described in WO 2007/111968. However, the invention described in WO 2007/111968 solves the opposite problem to the present solution, ie targeted silencing of specific genes. None of the aforementioned patent documents directly addresses the issue of protection against methylation and / or silencing of gene expression.

SUMMARY OF THE INVENTION

We have previously observed that an RSV offense is resistant to methylation and silencing when a CpG island from the mouse adenosine phosphoribosyltransferase (aprt) gene is adjacent (Hejnar et al.,

2001). This discovery is the basis of the present invention.

The aim was to construct a generally usable regulatory gene element that would prevent the silencing and methylation of the retroviral vector, thus enabling the use of, in particular, ASLV but also other retroviral vectors. The object of the invention was achieved in that the retroviral LTR was enriched in one or more of the central sequences (IEs) of the CpG island of the Syrian hamster aprt gene [Mesocrícetus auratus]. The regulatory gene element thus formed is able to ensure expression of the retroviral transduced transgene and effectively protect the integrated vector from silencing and sequential methylation.

We have found and described the surprising ability of the central sequence (IE) of the CpG island of the hamster aprt gene (Siegfried et al., 1999) to stabilize long-term expression of a retroviral vector and protect it from transcriptional silencing. In a study where they inserted this central sequence into RSV LTRs and prepared vectors showing stable and position independent expression of the reporter gene, they demonstrated that the modified LTR modification can therefore be used to construct vectors with prolonged transcriptional activity, particularly retroviral vectors, most preferably ASLV vectors. useful for gene therapy.

The conclusions of the study, which is described in more detail in the examples below, demonstrate that insertion of 120 bp IE into a specific LTR site will ensure efficient transcription of ASLV vectors and overcome repressive epigenetic effects in non-permissive mammalian cells. The best protective effect was found in a LTR with two IEs in tandem inserted between the promoter and enhancer sequences in an anti-sense orientation. None of the 19 cell clones analyzed transduced with this modification of the reporter vector showed signs of significant transcriptional silencing of the vector even after thirteen weeks of culture. Thus, the optimized vector construction can be used as a new strategy to improve retroviral vectors for efficient gene transfer and gene therapy.

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Accordingly, the present invention provides a regulatory gene element comprising a promoter / enhancer protected from DNA methylation and silencing of gene expression, the element comprising a modified retroviral LTR wherein one or two central IE sequences in any orientation are inserted into any LTR site, the IE having the sequence shown herein as SEQ ID NO. The skilled artisan will appreciate that the LTR DNA sequence may be isolated from a natural source (retrovirus) or may be synthetically prepared by methods well known in the art. The person skilled in the art is also aware of what sequence properties are functionally equivalent to the central IE sequence. The functional equivalent of the central IE sequence may be a sequence originating from a CpG island and / or having CpG island sequence properties as generally known in the art. One of ordinary skill in the art will be able to identify and isolate such an equivalent sequence from a natural source or prepare it synthetically, and utilize such a sequence to prepare a regulatory gene element containing a promoter / enhancer protected from DNA methylation and silencing gene expression. Thus, when the term CpG island central sequence (IE) or only the central sequence (IE), or abbreviated only IE, is used in the following description and claims, this term also includes an equivalent sequence, i.e. sequence originating from CpG island and / or having sequence properties Island CpGs, as known to those skilled in the art, which perform the same function in the LTR promoter as the described IE sequence (SEQ ID NO: 2).

The invention further provides said regulatory gene element, wherein at least one IE sequence is inserted at a position between the promoter sequence and the enhancer.

Further, the present invention provides a regulatory gene element as described above comprising a modified Rous sarcoma virus LTR sequence with an inserted tandem IE sequence, preferably all orientation, more preferably vanti-sense orientation, most preferably vanti-sense orientation at position -89 from the start of transcription.

The invention also provides a vector comprising at least one of the above-mentioned regulatory gene element and at least one gene to be expressed in a target cell, further terminator sequences and optionally other sequences, all sequences operably linked such that transcription of the gene to be expressed in the target cell, is under the control of a regulatory gene element.

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Further, the present invention provides the above-described vector, which is a retroviral vector, most preferably

ASLV vector.

A further object of the invention is a method of preventing promoter / enhancer methylation and transcriptional silencing of a gene, comprising the step of modifying a promoter / enhancer region in a vector containing a retroviral LTR containing a promoter / enhancer operably linked to the gene to be expressed in the host cell. such that one or two central IE sequences in any orientation are inserted into any ITR site, the IE having the sequence shown herein as SEQ ID NO. No. 2.

Furthermore, the invention also encompasses the method of the preceding point, wherein at least one IE sequence is inserted at a position between the promoter and enhancer sequences.

The invention also relates to a method as described above, wherein the Rous sarcoma virus LTR sequence is modified by inserting a tandem IE sequence, preferably in sense orientation, more preferably vanti-sense orientation, most preferably in anti-sense orientation at position -89 from the start of transcription.

Another object of the invention is a vertebrate cell, preferably a mammal, comprising a vector of the invention.

A subject of the invention is also a transgenic non-human organism comprising a cell according to the preceding paragraph.

The present invention is explained in more detail by the following examples, with reference to the accompanying drawings.

Description of the drawings

Giant. 1: Schematic representation of retroviral vectors used (A) The RNIG and MNIG base vectors containing the neo ^r and EGFP genes driven by the 5 'LTR of Rous sarcoma virus (RSV) or mouse leukemia virus (MLV), respectively. PBS refers to the primer binding site; ψ * is the MLV encapsidation signal; IRES refers to the internal ribosome entry site; PPT is a polypurine stretch.

(B) Nucleotide sequence of the central sequence (IE) of the CpG island of the hamster adenosine phosphoribosyltransferase (aprt) gene. Spi binding sites are indicated by boxes, CpG dinucleotides are shown in bold. Three base substitutions at each Spi binding site that reveal the ability to bind Spi are shown.

(C) Modification of LTRs in RNIG vector. A single or double IE was inserted into the indicated restriction sites of the sense and / or anti-sense orientation. The orientation of the IE is indicated by an arrow. The bicistronic coding regions and the 3'LTR are not shown.

Giant. 2: FACS analysis of GFP expression from a vector with modified and unmodified LTRs

NIL-2, HEK 293 and QT6 cell lines transduced with the vectors RNIG and RNIG2-2IE were selected using G418 and then analyzed 57 days after removal of the selective antibiotic by FACS. One clone from each cell line is shown with each of the two vectors. The relative GFP fluorescence versus cell number is plotted in the histograms and the percentage of GFP-positive cells is also shown. The range of GFP positivity is shown as a horizontal line.

Fig. 3: Stability of GFP expression from retroviral vectors with modified and unmodified LTRs during long-term cultivation of transduced cells

NIL-2 cells were infected with retroviral vectors modified by inserting IE (A) into the U5 region of the LTR, (B) between the LTR promoter and enhancer, and (C) at the beginning of the U3 region. Transduced cells were selected with G418 for 15 days, the percentage of GFP-positive cells was quantified by FACS at regular intervals after selection was removed, and the cells were cultured without selection. Each point represents the average of three culture dishes. (D, E) NIL-2 cells were infected with modified and unmodified vectors, after 7 days of selection individual clones of G418-resistant cells were isolated and cultured separately without selection. The time course of vector silencing was monitored by FACS at regular intervals and the percentage of GFP-positive cells is shown as the average of all cell clones transduced with the same vector. (E) Stability of GFP expression after removal of selection in 19 individual cell clones transduced with RNIG2-2IE vector. Graphs of 17 non-silenced clones were pooled into one. (D) Stability of GFP expression in nine individual cell clones transduced with the control RNIG vector. (F) Stability of expression of vectors modified by introducing IE with mutated Spi binding sites. The population of NIL-2 cells was transduced with vectors, selected using G418, and individual clones were isolated and cultured separately. Each curve represents the average percentage of GFP-positive cells of all cell clones transduced with the same vector construct.

Giant. 4: Reactivation of mutated vectors

NIL-2 cells were infected with vectors susceptible to silencing RNIG and RNIG3-MIE with mutated Sp1 binding sites. Cells were selected with antibiotic G418 and removed after six days. After 18 days of culture, GFP-negative cells were sorted by FACS. Each GFP-negative population was then divided into two subpopulations six days after sorting: one was then treated with 5-azaC and TSA for four days and the proportion of GFP-expressing cells was evaluated. The population of untreated GFP-negative cells was further cultured and treatment was repeated at regular intervals. Each experiment was performed in triplicate.

Giant. 5: DNA methylation of integrated vectors

The methylation status of CpG in LTR integrated vectors was tested by the bisulfite method.

(A) CpG methylation status in the 5'LTR sequence of silencing-prone vectors RNIG and RNIG3-IE. NIL-2 cells transduced with vectors were used to isolate the DNA nine weeks after removal of the selection, the DNA was examined by the bisulfite method. Each ring line represents one independent LTR sequence cloned from the PCR product performed with bisulfite treated DNA. The percentages of GFP-positive cells and methylated CpGs are shown.

(B) CpG methylation status in the 5'LTR sequence of RNIG2-2IE vector in two cell clones. The analysis was performed using the bisulfite technique, see a.

(C) Methyl status of CpG pairs in the 5'LTR sequence of silencing-prone vectors RNIG and RNIG3-MIE. NIL-2 cells transduced with vectors and enriched in GFP-negative cells by FACS three weeks after removal of the selection were harvested and subjected to sequencing by the bisulfite method. Each ring line represents one independent LTR sequence of the PCR product obtained from bisulfite treated DNA. The percentages of GFP-positive cells and methylated CpGs are shown. Methylated CpGs are shown as solid circles, unmethylated CpGs are shown as open circles.

DETAILED DESCRIPTION OF THE INVENTION

Example 1: Study of the effect of the central IE sequence on the expression of retroviral vectors

In the study underlying the present invention, we investigated the effect of the short central sequence (IE) of the Crt island of the aprt gene of the Syrian hamster (Mesocricetus aurotus) on the expression of RSV and MLV-based retroviral vectors and their resistance to transcriptional silencing.

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The central sequence of the CpG island (IE) stabilizes the long-term expression of the LTV-driven retroviral vector from RSV

We constructed retroviral vectors RNIG and MNIG with a neomycin resistance gene (neo ^f ) and an enhanced green fluorescence (GFP) protein gene under the control of the LTRs of RSV (RNIG) and MLV (MNIG). Both genes were expressed from one bicistronic mRNA via an internal ribosome entry site (IRES) from the encephalomyocarditis virus (Fig. 1a). The LTV of RSV contains a highly potent promoter but is prone to CpG methylation and transcriptional silencing upon infection of mammalian cells (Searle et al., 1984, Hejnar et al., 1994).

The structure of the RSV LTR (Prague strain RSV-Pr-C) is as follows (SEQ ID NO: 1):

tgtagtcgtacgcaatactcctg | tagtcttgc | aacatgcttatgtaacgatgagttnagcaatatgccttacaaggaaagaaaaggc

ccactRaattccgcatcgcagagatattgtatttaagtgcctagctcgatacaataaacgccotfflqccqftcaccocgttgetgtgc acctgggttgatggccggaccgtcgattccctaacgattgcgaacacctgaatgaagcagaaggcttca, wherein the frame are indicated by two enhancer motifs, bold and underlined are indicated promoter sequence attgg (inverted CCAAT) and tatttaa (TATAbox) italics and underlined is indicated repetitive segment R which separates the segment U3 (left) and U5 (right) and whose first nucleotide is the start of transcription.

A sequence of IE aprt CpG hamster island characterized previously as an effective protective motif was selected to protect the RSV LTR against silencing (Siegfried et al., 1999). IE represents a 120 bp sequence that includes seven CpG dinucleotides and two high affinity Spi binding sites (Fig. 1b) and is responsible for most of the properties of the CpG island. The sequence of said IE is as follows (SEQ ID NO: 2):

tccagcaaatgctttacttcctgccaaaagccagcctccc ca caacccactctcccaeaggccc§ccc§tc (ccccccctcc§g wherein CpG dinucleotides are shown in bold and boxed, the Factor Spi binding sites are underlined.

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The LTV sequence of the RSV was modified as follows: One IE was inserted in both orientations at three different positions of the RSV LTR: I) in the U5 region downstream of the promoter and transcriptional origin, II) in the middle of the U3 region between enhancer and promoter and III) U3 regions upstream of enhancer. In another variant of the LTR modification, two IEs were inserted in the anti-sense orientation between the enhancer and the promoter. The U5 region was used at the 5'LTR and U3 at the 3'LTR, so after reverse transcription of the corresponding RNA, all modifications appeared in both LTRs of the resulting provirus. The entire set of modified RSV LTRs with the appropriate designation of the corresponding retroviral vector is shown in Figure 1c.

For long-term expression stability studies, wild-type and modified vectors were packaged in GP293 cells and transduced into two avian cell lines, chicken DF-1 and quail QT6, and two mammalian cell lines, hamster NIL-2 and human HEK 293 Cells containing transcriptional transduced neomycin resistance genes were selected with antibiotic 6418 and GFP expression was monitored at regular intervals after removal of G418 by activated fluorescence microscopy and cytometry (FACS). All vectors with wild-type LTRs or modified LTRs showed very stable expression in DF1 during in vitro culture. Few GFP-negative cells appeared after two months of culture without selection pressure (data not shown). Similar stability of GFP expression was also observed in QT6 cells transduced with the vectors RNIG, RNIG2-2IE and MNIG. After transduction of each of the three vectors, six single-cell clones of G418-resistant cells were isolated and cultured separately for more than two months. All clones showed very stable expression often without any indication of silencing (data not shown). Only one QT6 clone transduced with a vector with unmodified RSV LTR was progressively silenced, and after 70 days of culture (without selection), 22% of GFP cells were negative.

Significantly, different behavior of integrated vectors was observed after transduction of mammalian cells. The RNIG and MNIG vectors were sequentially silenced in NIL-2 cells, while the vectors inserted with the central IE sequence showed a different degree of protection from transcriptional silencing (Figs. 2, 3A-C). The insertion of one or two IEs between promoter and enhancer almost completely stabilized long-term expression of RNIG vectors, regardless of ΙΕ orientation (Fig. 2, 3B). Due to the significant variability between cell cultures transduced with the same modified vector, several G418-resistant NIL-2 clones were isolated after infection with individual vectors and GFP expression was observed over time. Different numbers of clones were checked, six clones with RNIG2 + IE vector and up to 19 clones with RNIG2-2IE vector. Data are shown individually for wild-type RNIG and RNIG2-2IE vectors and cumulatively for the remaining vectors. The RNIG vector was progressively silenced in most cell clones. Of the nine isolated clones, only two were non-variegated and showed stable expression of GFP (Fig. 3D), presumably due to integration into a particular site in the genome of the host cell that promoted efficient transcription of the integrated retrovirus. In contrast, almost no silencing was observed in clones carrying the RNIG2-2IE vector modified by introducing a double IE at position -89. 19 clones of NIL-2 cells transduced with this vector were analyzed and very low silencing of GFP expression was observed in only two of them. After 91 days of culture 2% and 8% GFP negative cells were shown. The other 17 clones showed no indication of any silencing (Fig. 3E).

All these experiments were repeated and confirmed on the HEK 293 human cell line. The data on silencing of the protective effect of IE against silencing fully corresponds to that obtained on NIL-2 cells. As a rule, the only difference was an even slower silencing pattern in the HEK 293 cell line. GFP fluorescence intensity was essentially the same for NIL-2 and HEK 293 cells, but higher for QT6 cells. This agrees with the expected higher level of RSV LTR-directed transcription in avian cells. Insertion of IE, both single and tandem, between enhancer and promoter did not increase GFP fluorescence (Fig. 2), meaning that transcription and titers of modified vectors were unaffected.

Insertion of one IE, preferably two IEs in tandem, between the enhancer and the promoter effectively prevented silencing of transcription.

The skilled artisan will appreciate that the LTR DNA sequence may be isolated from a natural source (retrovirus) or may be synthetically prepared by methods well known in the art. The person skilled in the art is also aware of the properties of a sequence that is functionally equivalent to the central IE sequence. The functional equivalent of the central IE sequence may be a sequence originating from a CpG island and / or having CpG island sequence properties such as t

generally known in the art. One of ordinary skill in the art will be able to identify and isolate such an equivalent sequence from a natural source or prepare it synthetically, and utilize such a sequence to prepare a regulatory gene element containing a promoter / enhancer protected from DNA methylation and gene expression silencing. Thus, when the term CpG Island Central Sequence (IE) is used in the following description and claims, this term also includes an equivalent sequence, i.e., a sequence originating from a CpG island and / or having CpG island sequence characteristics as known to those skilled in the LTR. promoter the same function.

Spi binding sites are important for the protective role of the IE CpG island

The IE used in these experiments contains two Spi binding sites. Point mutations have been introduced at the Spi sites that have abolished the ability of the DNA sequence to bind the Spi protein. The vectors RNIG2-IE, RNIG3 + IE, and RNIG3-IE mutated in this manner were termed RNIG2-MIE, RNIG3 + MIE, and RNIG3-MIE, respectively. All Spl-mutated vectors showed a significantly reduced protective effect on GFP expression in NIL-2 cells as compared to their non-mutated counterparts. The mutated RNIG3 + MIE and RNIG3-MIE vectors were silenced even faster than the original vectors without any inserted IE (Fig. 3F). In DF-1 and QT6 cells, GFP expression from the RNIG3 MIE vector was stable and showed no silencing (data not shown). It has been shown that one intact Spi site is essentially necessary for an effective IE function to prevent silencing (with the reservations set forth in the discussion in the Discussion section below).

Reactivation of silenced vectors with 5-azacytidine and trichostatin A

We investigated the possibility of reversing transcriptional suppression of integrated provirus DNA by a methyltransferase inhibitor, 5-azacytidine (5azaC) and / or a histone deacetylase inhibitor, trichostatin A (TSA). GFP-negative cells of NIL-2 silencing-prone clones transduced with RNIG and RNIG3-MIE vectors were cytometrically sorted 11 days after G418 harvest and cell cultures were treated at regular time intervals with the indicated inhibitors, either alone or in combination. The increase in the percentage of GFP-positive cells was assessed by FACS analysis. Both 5-azaC and TSA used separately reactivated GFP expression (data not shown), but the combination of both inhibitors gave the strongest effect (Fig. 4).

Most of the silenced vectors cultured 21 days after G418 harvest were reactivated with 5-azaC and TSA, however the effect of the inhibitors gradually decreased and only 2% of the vectors were reactivated after 112 days of culture without selection (Fig. 4).

DNA methylation analysis of Integrated vectors

Since transcriptional silencing of genes or proviruses is usually due to DNA methylation in the promoter region, the CpG methylation profiles of LTR vectors that were silenced or transcriptionally active were analyzed and compared. Two clonal NIL-2 cell cultures transduced with the RNIG vector, one high and the other with a very low percentage of GFP-positive cells, and one clonal cell culture transduced with the RNIG3-IE vector with 30% GFP-positive cells were selected for this analysis. These clones were tested approximately nine weeks after G418 selection of transduced cells. Bisulfite sequencing of the vectors showed almost completely methylated 5'LTR sequences in a culture with a silenced RNIG provirus. In contrast, almost no CpG methylation was detected in cultures without silencing the RNIG vector {FIG. 5a). A clone with 30% GFP-positive cells was among the reported values in terms of methylation of the 5 * LTR RNIG3-IE vector. There was no apparent methylation profile and methylated CpG dinucleotides were observed throughout the LTR sequence. Clones transduced with the RNIG2-2IE vector that showed no silencing of integrated vectors also had a negligible 5'LTR methylation level. IE sequences were also inserted without methylation (Fig. 5B).

In order to monitor the early phase of vector silencing, CpG methylation was examined in rare GFP-negative cells that appeared soon after the selection of transduced cells. Two NIL-2 cell cultures transduced with the RNIG and RNIG3-MIE vectors were selected from these cells by FACS after 21 days of culture without selective pressure. Since it is difficult to separate weakly expressing cells, the obtained cell cultures were enriched in GFP-negative cells but also contained 15% and 28% positive cells, respectively (Fig. 5C). The CpG methylation density in the LTR sequence was 28% and 19%, respectively, much lower than the nine

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- * 1 4 .4 11 week old RNIG3IE transduced cell culture containing a comparable percentage of silenced cells (Fig. 5C).

Based on the above findings, it can be concluded that silencing of the integrated provirus is associated with CpG methylation of the promoter sequence, but there are other silencing mechanisms that precede strong methylation of the proviral LTRs.

In this experiment, the inventors have shown that the insertion of at least one IE protects the retroviral LTR from both types of transcriptional suppression.

Discussion

This section discusses the results obtained for the sake of study integrity and scientific integrity. Regardless of the various theories, the results fully support the claimed solution and do not in any way call into question the possibilities of its practical application, which have been described.

There are two basic strategies to “combat the silencing of retroviral vectors derived from gammaretroviruses and lentiviruses. The first is the elimination of the so-called silencers defined eg in the LTR and the MLV primer binding site. Multiple point mutations of all CpGs in the LTR region also stabilize expression of MLV vectors in embryonic stem cells (Swindle et al., 2004). Alternatively, DNA segments were used to bind to the nuclear matrix or "isolators to prevent the positional effects of adjacent cell silencers (Rivella et al., 2000; Yannaki et al. 2002; Zhang et al., 2007).

Previous experiments with the CpG island of the mouse aprt gene (Hejnar et al., 2001) adjacent to the RSV reporter provirus showed that protection against silencing and methylation could also be due to some isolation effects. We have found that the introduction of a CpG island protects the downstream lying enhancer / promoter in an orientation-dependent manner without any increase in promoter strength.

In the experiments described in the present application, a short sequence (IE) from the CpG of aprt hamster island (Siegfried et al., 1999) was used and inserted into retroviral LTRs. This IE sequence shows the full protective effect of the entire CpG island, but differs in several features. The observed stabilization of reporter gene expression was significantly dependent on the IE position in the LTR. The IE inserted at the beginning of the U3 region had less effect, but insertion between the viral enhancers and the promoter resulted in very effective protection against silencing.

We did not have accurate data on the strength of LTR promoters modified by IE insertion, but estimation of reporter expression level based on FACS analysis (Fig. 2) shows that there are negligible differences between the RSV LTR variants. These data contradict the effect of isolation elements defined as sequences capable of preventing external enhancers from influencing promoters. The extent of the antimethylation effect is limited to approximately 150 bp from IE (Hejnar et al., 2001). This could explain the weak or no protective effect of the IE inserted upstream of the enhancer and the best protective effect of the IE inserted between the enhancer and the promoter. Both transcriptional regulators are thus within the range of the IE effect. The position 89 bp upstream of the transcription start closest to the IE position in the hamster aprt gene, 107 bp upstream of the start of transcription. This distance is likely to be favorable for maintaining a transcriptionally effective promoter. In contrast to the complete CpG island of the mouse, the effect of IE orientation is only weak.

The role of Spi binding sites in the antlmethylation capacity of CpG islands has been described (Macleod et al., 1994; Brandeis et al., 1994), but the exact mechanism of their action is unknown. We tested the effect of Spi mutations in three modified LTRs. In all three vectors, the mutation of the Spi site significantly increased provirus silencing during long-term cultivation, thereby abolishing the protective effect of the inserted IE. In particular, the RNIG3-MIE vector was silenced even more efficiently than an unmodified LTR vector. This was probably due to the accumulation of CpG dinucleotides that are, regardless of the Spi site dependence, the target sites of DNA methyltransferases. On the other hand, the most stable RNIG2-IE vector retained part of the silencing protection capabilities even with the mutated Spi site. This demonstrates that the Spi binding site contributes to the protective ability of IE against silencing, but is not the only cis-acting element.

Reactivation experiments led to the finding that vector silencing was associated with DNA methylation and / or histone deacetylation, as described in detail in experiments with ientiviral vectors (He et al., 2005).

Effective silencing of ASLV-based vectors has been described in rodent cells. To verify the relevance of these results in human cells, we conducted experiments in parallel with the HEK 293 cell line. The results obtained are essentially the same as those obtained for

NIL-2 cells for all 5'LTR modified vectors, but the silencing of the wild-type RNIG vector was slower and, therefore, the protective effects introduced by IE were less pronounced.

Transcriptional silencing is a major obstacle to the use of retroviral and lentiviral vectors in gene transfer and gene therapy (Ellis, 2005). The anti-methylation protection strategy of ASLV-based vectors using the central sequence of the CpG island shows that it is possible to prepare a fully silent-resistant retroviral vector. The vector or method of the invention therefore appears to be immediately applicable at any time, for example when RCAS vectors are used in mammalian cells, and may be the basis of an optimized vector construction.

Example 2: Materials and procedures used in the study

Design of retroviral vectors

Plasmids pLPCX, pLXRN, plRES2-EGFP (all Clontech, Mountain View, CA) and pH19KE were used to prepare the series of constructs used in this experiment. Neomycin resistance gene (ne <f) such as 1.419 bp Sg / L | - The EcoRV fragment from plasmid pLXRN was inserted into the multiple cloning site p (RES2-EGFP digested with flg / II and Smol to obtain pN-IRES-G. The pRMR construct was constructed by ligating a 3.445 bp SsfEII-So / I pH19KE fragment carrying LTR RSV and 2.548 bp PshM-PvM internal fragment of pLPCX The cassette containing neo ^r , IRES and EGFP, 2.864 bp Eco47II-Hpa \ fragment pN-IRES-G was then ligated to 4.579 bp Smol and XhoL major fragment pRMR Resulting retroviral vector pRNIG (Fig. 1A) was used to construct all insertion variants of the RSV LTR The pMNIG vector was prepared by ligating the 3.509 bp SstE II - Cla I cassette neo ^r , IRES and EGFP from pRNIG and the 4.458 bp SstEII - C / ol LTR fragment from pLPCX. The 'and 3' LTR sequences in pLPCX are derived from the murine leukemia virus Moloney virus and its replication-defective derivative of the murine murine sarcoma virus Moloney virus, respectively. Minor differences in the sequences of these LTRs made it possible to distinguish the cloned plasmid retroviral virus. DNA from proviral DNA that has undergone reverse transcription.

t I

Three unique restriction sites were introduced into the LTR sequences of pRNIG by in vitro mutagenesis (see below). The B'I / WI site at position -226 upstream of the transcriptional origin and the SstZ17I site at position -89 were inserted into the 3'LTR and the Hpa I site at position +27 was inserted into the 5'LTR. Unique restriction sites were used to insert the central IE sequence. PRNIG1 + IE and pRNIG1-IE were prepared by inserting IE into the Hpa I site in the sense and anti-sense orientation, respectively. PRNIG2 or pRNIG3 variants were prepared analogously by inserting IE into the 8stZ171 or BsiWI site, respectively. The PRNIG2-2IE variant was prepared analogously by inserting two copies of IE into the SstZ171 site in the anti-sense orientation (Fig. 1C). The IE inserted vectors containing the mutated Sp1 binding sites (see below) were named RNIG3 + MIE, RNIG3-MIE, and RNIG2-MIE. The IE insertion and orientation sites were confirmed by DNA sequencing.

Preparation of IE

The central sequence (IE) of the CpG island of the aprt gene (45) was prepared by "gene assembly PCR" using eight oligonucleotides:

1F: 5'-agtcgtatactccagcaaatgcgttacttcctgcc-3 ',

2F: 5'-aaaagccagcctccccgcaacccac-3 '

3F: 5'-tctcccagaggccccgccccgtccc-3 '

4F: 5'-gccccctcccggcctctcctcgtgctgg-3 ',

IR: 5'-tgcggggaggctggcttttggcagg-3 ',

2R: 5'-gggcggggcctctgggagagtgggt-3 ',

3R: S'-cgaggagaggccgggagggggcgggacg-3 'a

4R: 5'-atgcgtatactccttagggagcgatccagca-3 '.

These oligonucleotides were combined into pairs, 1F + 1R, 2F + 2R, 3F + 3R and 4F + 4R in four reactions, 1-4, respectively. The cycling conditions were as follows: 96 ° C for 2 minutes, 4 cycles of 95 ° C for 40 s, 70 ° C for 10 s, 0.3 ° C / s drop to 25 ° C and 72 ° C for 1 minute, and 35 cycles: 95 ° C for 30 sec, 54 ° C for 30 sec and 72 ° C for 30 sec, final extension at 72 ° C for 3 minutes. Reaction products 1 and 2 were mixed and PCR prepared with primers 1F and 2R. Reactions 3 and 4 were performed analogously to primers 3F and 4R. The PCR conditions were as follows: 96C for 2 minutes and 35 cycles: 95'C for 30s, 50C for 30s and 72'C for 40s, final extension at 72'C for 5 minutes. The products of the two reactions were mixed into one reaction and PCR was performed under the conditions used in the previous PCR with primers 1F and 4R, which reaction yielded a 142 bp product with a S 2 Z 17 I restriction site at each end. To prepare IE with SsiWI restriction sites, the final product was PCRed with primers 1Fb: 5'-agtccgtacgtccagcaaatgcgttacttcctgc-3 'and

4Rb: 5'-agtccgtacgtccttagggagcgatccagc-3 '.

Site-directed mutagenesis

All site-directed mutagenesis experiments were performed using a Transformer site-directed mutagenesis kit (Clontech, Mount View, CA) according to the manufacturer's protocol. The synthesized IE fragment was cloned into the pGEM-T Easy vector (Promega, Madison, WI).

A mutagenic primer was used to mutate the Spi binding sites:

5'-ctcccagaggcccattcccgtcctttcccctcccggc-3 ', which also served as a selection primer. Selection was performed with the restriction enzyme Drali. The mutagenic primer: 5'ggaaatgtagtcgtacgcaatactcctg-3 'was used to introduce the unique cloning site into the pRNIG construct, the mutagenic primer:

5'-cttattaggaaggtatacagacgggtc-3 'for introduction of BstZ17l site and mutagenic primer:

4

- acattggtgttaacctgggttg-3 'for introducing Hpal space.

Selection of vectors with new sites was carried out alternately using two selection primers, the Scol / Bg / II selection primer:

5'-gtgactggtgagatctcaaccaag-3 'and Sg / II / Scal reselection primer:

'-gtgactggtgagtactcaaccaag-3'.

Cell culture

The GP293 packaging line (Clontech, Mountain View, CA) was maintained in D-MEM / F12 medium (Sigma, St. Louis, MO) with the addition of 5% newborn calf serum, 5% fetal calf serum (both Gibco-BRL, Gaithersburg, MD) and penicillin / streptomycin (100 mg / ml each). The human germline kidney cell line HEK293, the hamster fibroblastoid line NIL-2, the methylcholanthrenic quail cell line QT6, and the chicken fibroblast cell line DF1 were maintained in D-MEM / F12 medium supplemented with 5% newborn calf serum, 2% fetal calf serum both Gibco-BRL, Gaithersburg, MD) and penicillin / streptomycin (100 mg / ml each). Tissue cultures were cultured at 37'C in an atmosphere of 3% CO2. In reactivation experiments, 5-AzaC and TSA were used to treat cells with 4 μΜ 5-azaC (Sigma) and 0.5 μΜ or 1 μΜ TSA (Sigma) for 4 days.

Propagation of vectors

The MNIG and RNIG vectors and their modifications were propagated by transfecting plasmid DNA containing proviral forms of reporter vectors together with plasmid pVSV-G (Clontech, Mountain View, CA) into GP293 cells. GP293 cells at 1.5 x 10 ⁷ were seeded in 140 mm Petri dishes. After 24 hours, cells were cotransfected with 10 µg pVSV-G and 50 µg vector plasmid. Co-transfection was performed by precipitation with phosphate »

calcium. Culture medium containing vector particles was harvested 1, 2 and 3 days after transfection. The collected viral solutions were centrifuged at 200 xg for 10 minutes at 4 ° C. The supernatant was collected and centrifuged at 24,000 rpm for 2 hours and 30 minutes at 4 ° C in a SW28 rotor, Beckman OptimalOO (Beckman, Fullerton, CA). The pellet was resuspended in culture medium with 10% calf serum of newborn calves, frozen and stored at -80 ° C. Titration of infectious viral particles was performed by serial dilution of the viral solution followed by infection of DF1 cells. In repeated experiments, vectors with modified and unmodified LTRs achieved similar titers in the range of 2 x 10 ⁴ - 10 ⁵ IU / ml. Twenty-four hours after infection, 400 µg / ml G418 (Sigma, St. Louis, MO) was added and cells were selected for 15 days. After selection, the number of G418-resistant colonies was evaluated.

Cell transduction and FACS analysis

Cells were plated in Petri dishes at 5 x 10 ⁵ cells per 60 mm dish and after 5 hours 200 µΙ of virus suspension with 15 µg / ml polybrene was added and allowed to adsorb for 40 minutes at room temperature. After adsorption fresh medium was added to 4 ml and cells were placed at 37 ° C and 3% CO 2. Twenty-four hours after infection, selection was initiated with 400 µg / ml G418 (Sigma) and the selection medium was changed every two or three days. After 15 days of selection, G418 was harvested. At weekly intervals, cell cultures were analyzed using an LSR II cytometer (Becton Dickinson, San Jose, CA) and GFP ⁺ cell frequency was evaluated. At specific intervals, cultures were sorted using a FACSVantage SE (Becton-Dickinson) according to the presence or absence of GFP expression. For clonal FACS analysis, cell clones were obtained by limiting dilution of transduced cell cultures.

Analysis of methylation

Genomic DNA isolated from infected cells was treated with sodium metabisulfite using the EpiTect kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. The semi-nested upper strand PCR was performed with primers complementary to the U3 region of the RSV LTR and the leader (Fig. 5b) containing up to one CpG in the LTR. The primer sequences were as follows:

5'-gttttataaggaaagaaaag-3 '(top),

5'-aacccccaaataaaaaacccc-3 '(lower-inner) a

5'-aaacaaaaatctccaaatcc-3 '(lower-outer),

PCR reactions were performed with 200 ng of DNA for 25 cycles: 95 ° C for 1 minute, 58 ^and C for 2 minutes and 72 ° C for 90 seconds. The PCR products were cloned into the pGEM-T Easy vector (Promega) and sequenced using universal pUC / M13 primers.

List of references cited

Patent documents

U.S. 7,232,654

WO 91/02805

EP 0 801 575

2006/0153810

US 5,814,493

WO 01/48229

WO 2007/111968

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Claims (10)

PATENT CLAIMS PV 2OOS · U5 75 221 CLAIMS 1. A regulatory gene element comprising a promoter / enhancer protected from DNA methylation and silencing of gene expression, the element comprising a modified retroviral LTR sequence wherein one or two central IE sequences in any orientation are inserted into any LTR site, the IE having the sequence shown herein as SEQ ID NO. No. 2. The regulatory gene element of claim 1, wherein at least one central IE sequence is inserted at a position between the promoter sequence and the enhancer. The regulatory gene element according to claim 1 or 2, comprising a modified Rous sarcoma virus LTR sequence with the inserted tandem IE sequence, preferably in sense orientation, more preferably vanti-sense orientation, most preferably vanti-sense orientation at position -89 from the start of transcription. A vector comprising at least one regulatory gene element according to any one of claims 1 to 3 and at least one gene to be expressed in the target cell, further terminator sequences and optionally other sequences, all sequences being operably linked such that gene transcription, which is to be expressed in the target cell is under the control of a regulatory gene element. The vector according to claim 4, which is a retroviral vector, preferably an ASLV-based vector. 6. A method of preventing promoter / enhancer methylation and transcriptional gene silencing comprising the step of modifying a promoter region in a vector comprising a retroviral LTR containing a promoter / enhancer operably linked to a gene to be expressed in a host cell. / amplifier such that one or two central IE sequences in any orientation are inserted at any LTR site, the IE having the sequence shown herein as SEQ ID NO. No. 2. 7. The method of claim 6, wherein at least one IE sequence is inserted at a position between the promoter and enhancer sequences. The method of claim 6 or 7, wherein the Rous sarcoma virus LTR sequence is modified by inserting a tandem IE sequence, preferably in all orientation, more preferably in anti-sense orientation, most preferably in anti-sense orientation at position -89 from the start of transcription. A vertebrate cell, preferably a mammal, comprising the vector of claim 4 or 5. A non-human transgenic organism comprising the cell of claim 9.