SE518759C2 - Vectors resistant to methylation - Google Patents

Abstract

The invention relates to vectors produced in a donor host cell, which upon transfer into a receiver host cell maintain the desired expression of the nucleotide sequences which are located within the vector. The maintenance of the desired expression is achieved due to that the vector at least partly remains unmethylated within the receiver host cell. The donor host cell being different as compared to the receiver host cell and the receiver host cell being capable of methylating DNA. To prevent methylation, cytosines in a CpG motif in the nucleotide sequence to be transferred are replaced with cytosine analogues resistant to methylation. The invention also relates to methods for the production of such vectors and the use of the vectors in industry as well as in medicine.

Description

518 759 9.

DNA affects the level of expression of the particular DNA. The introduced DNA can u ø o ø n I - - | - u u - - v. is also incorporated into the chromosomal DNA of a dividing cell by recombination.

If this particular DNA is methylated prior to incorporation with the chromosomal DNA and cell division, it will probably also be methylated during subsequent cell divisions. If a high level of expression is desired, the methylation of DNA in expression vectors is a problem.

A similar problem also occurs when using vectors in the treatment of disorders and in vaccination in medicine. For example, the DNA vaccine is usually in the form of a plasmid DNA expression vector made in bacteria and then purified and given to muscles and skin. DNA vaccines have shown efficacy against a large number of viral, bacterial and parasitic disorders in DJ unnodels.

However, the level of methylation of such DNA will affect the level of expression of antigenic proteins, which are necessary for the induction of immune responses to the antigen. The same problem applies to the use of DNA vectors in gene therapies.

One way to solve the methylation problem of nucleotide sequences upon transfer into a new host is to manipulate the genes and remove all CpG motifs without altering the polypeptides they encode. Such modified genes will not be methylated. The strategy for using such modified genes is well suited for genes with a shorter nucleotide sequence but cannot be applied to longer nucleotide sequences such as vectors or when there is a need for your different vectors.

In addition, the promoters used for gene expression are often rich in CpG motifs which cannot be removed without damaging the function of the promoter and thus the level of expression.

There is a need for stable optimized vectors that do not undergo methylation upon transfer into a eukaryotic cell for use in both industry and the art of medicine. By providing such vectors, the industry will have less trouble obtaining a constant and high expression of interesting genes in eukaryotic cells.

Furthermore, the art of medicine will provide improved vectors, vectors that do not undergo methylation upon transfer into a recipient host cell where the expression of the nucleotide sequences placed in the vectors is maintained, which is 518,759 u u u - | ~ u u o »I ~ v v | now useful both in vaccination and gene therapy as well as for all other treatments where expression vectors are used.

BRIEF DESCRIPTION OF THE INVENTION The invention relates to vectors that have been modified in such a way that methylation of the vector is significantly reduced upon transfer of the vector into a recipient host cell. Thereby, the expression of the nucleotide sequence of the vector is maintained at an acceptable level in a recipient host cell. A level that maintains the functions expected to be obtained from the nucleotide sequences and / or polypeptides encoded by the nucleotide sequences. The invention also relates to methods for producing said vectors and using the vectors in industry as well as in pharmaceuticals, eg treatment and / or diagnostics.

Accordingly, as a first object, the invention relates to a vector comprising a nucleotide sequence, wherein one or two cytosines in at least one CpG motif are replaced by a cytosine equivalent that is resistant to methylation.

In another object, the invention relates to a donor host cell comprising a vector having a nucleotide sequence, wherein one or two cytosines in at least one CpG motif are replaced with a cytosine equivalent that is resistant to methylation.

For a further purpose, the invention relates to a nucleotide sequence which is part of a vector obtained from a donor host cell and / or a polypeptide made by a donor host cell.

For a further purpose, the invention relates to a pharmaceutical composition comprising a vector and / or a donor host cell and / or a nucleotide sequence and / or a polypeptide and a pharmaceutically acceptable diluent, carrier, adjuvant or excipients.

For yet another object, the invention relates to a method of reducing methylation of CpG motifs in a vector in a recipient host. The method comprises replacing at least one cytosine in a CpG motif with a cytosine equivalent.

For yet another purpose, the invention relates to equipment comprising a vector and / or donor host cell. In yet another object, the invention relates to a method of transferring a vector, a nucleotide sequence, a polypeptide or a pharmaceutical composition into a recipient host cell, the method being selected from the list consisting of electroporation, microprojectile shelling and liposome-mediated delivery.

For yet another object, the invention relates to a vector, a donor host cell, a nucleotide sequence, a polypeptide or a pharmaceutical composition for use in treatment and / or diagnostics.

For a final purpose, the invention relates to the use of a vector, a donor host cell, a nucleotide sequence, a polypeptide or a pharmaceutical composition for the manufacture of a medicament for use in treatment and / or diagnostics.

The invention provides completely new vectors, in which cytosine in CpG motifs has been replaced by a cytosine equivalent. The cytosine equivalent is resistant to methylation. The cytosine equivalent replaces at least the 5-position in the pyrimidine ring of the cytosine in the CpG motif. Such replacement leads to an improved vector which upon transfer into a recipient host cell, such as a eukaryotic cell, retains the activity / expression of the nucleotide sequence of the vector. Furthermore, the invention provides methods for producing such vectors and the use of such vectors in, for example, both industry and the art of medicine.

DESCRIPTION OF THE DRAWINGS The invention is illustrated with reference to the drawings, in which Fig. 1 shows the structure of 5-azadeoxycytidine.

Fig. 2 shows an example of a general structure of a cytidine derivative.

DETAILED DESCRIPTION OF THE INVENTION Definitions In the context of the present application and invention, the following definitions apply: The term “nucleotide sequence” is intended to mean a sequence of two or more nucleotides. The nucleotides may be of genomic DNA, cDNA, RNA, semisynthetic 518 759 B or synthetic origin or a mixture thereof. The term includes single- and double-stranded forms of DNA and RNA. u n ~ a «u o | u a ø ø v; - now The term “vector” is intended to mean a nucleotide sequence which is usually a circular duplex of DNA capable of propagating independently of chromosomal DNA to a number of copies in a host, i.e. having a copy start. The vector may also be a vector incorporated with the genome of the recipient host cell. The vector is maintained stably and / or produced in values during propagation, such as by using a selection marker in the vector. The vector can be a bacteriophage, plasmid, phagemid, viral vector, plant transformation vector, insect vector or YAC (yeast artificial chromosome).

The term "copy start" is intended to mean a nucleotide sequence in which the DNA synthesis for vector copying begins. Copy start can occur at one or platser your sites in the vector depending on the vector used, such as at a site in a plasmid vector or at fl your sites in an adeno vector. Copy start is commonly referred to as the start of copying (abbreviated ori site) in a plasmid vector.

The term "selection marker" is intended to mean, for example, a gene encoding a polypeptide which confers resistance to a drug, for example ampicillin, kanamycin, tetracycline, chloramphenicol, neomycin, hygromycin or methotrexate.

The term "control sequence" or "control sequences" is intended to mean nucleotide sequences involved in the control of an action reaction. This includes nucleotide sequences and / or proteins involved in the regulation, control or influence of the expression of structural genes or of the copying, selection or maintenance of a plasmid or viral vector. Examples include attenuators, switches, amplifiers, controllers, terminators and promoters.

The term "CpG motif" is intended to mean a double-stranded nucleotide sequence having a cytosine followed by a guanine, which are linked by a phosphate bond, i.e. the CpG motif has two cytosines, one placed in each strand of the double-stranded nucleotide sequence.

The term "methylation" or "methylated CpG motif" is intended to mean that cytosine is methylated on the pyrimidine ring, which usually occurs at the 5-position of the pyrimidine ring. One or more of the methylated CpG motifs in a nucleotide sequence may lead to a complete shutdown of the expression of the nucleotide sequence. If methylated CpG- n <v »c u v v u u v u | If motifs are located within a nucleotide sequence encoding a polypeptide or within other nucleotide sequences involved in the expression of a specific nucleotide sequence, such as a control sequence as defined above, a significant or complete reduction in the expression of said nucleotide sequence is obtained.

The term "resistant to methylation" is intended to mean the absence of methylation of one or both of the 5-positions in the pyridmidine rings present in the CpG motif. One or more of your CpG motifs may be unmethylated within a vector that is resistant to methylation. Resistance to methylation is achieved by replacing an existing cytosine with a cytosine equivalent.

The term "recipient host cell" is intended to mean a cell which is not the same as the donor host cell. Inside a recipient host cell, a vector is methylated in the CpG motifs, since the recipient host cell initiates methylation of CpG motifs in nucleotide sequences that are not equal to the nucleotide sequence of the recipient host cell. This can happen when the recipient host cell receives a vector that has been generated / multiplied in a donor host cell. The methylation of the vector leads to a reduced or complete closure of the vector.

The term “replaced with a cytosine equivalent” is intended to mean that an indistinguishable cytosine in a CpG motif as defined above is replaced by a cytosine equivalent.

The cytosine equivalent replaces at least the 5-position in the pyrimidine ring of cytosine.

The cytosine equivalent is resistant to methylation. However, the cytosine equivalent may partially or completely replace cytosine. Cytosine is replaced by a cytosine equivalent during copying of the vector.

The term "cytidine derivative" is intended to mean a cytidine derivative capable of replacing cytosine in a CpG motif without affecting the activity of the nucleotide sequence containing the replaced cytidine derivative. The activity of the nucleotide sequence may be the same as before cytosine was replaced by a cytidine derivative. For example, a nucleotide sequence encoding a polypeptide, even after replacement of one or more cytosines with cytidine derivatives, will still be active and express the polypeptide.

The cytidine derivative is a derivative which cannot undergo methylation at the 5-position of the pyrimidine ring present in the cytidine derivative. Examples of cytidine derivatives are shown in Figs. 1 and 2.

The term "retain expression" is intended to mean expression of the nucleotide sequence that encodes a polypeptide and contains one or more cytosine equivalents, i.e., retains the expression to at least one acceptable level. The level is higher than the unmethylated form of the nucleotide sequence. The retained expression may be within a range higher than the expression of the methylated form of the same nucleotide sequence or the expression may be as high as the expression of the unmethylated form of the same nucleotide sequence.

The term "donor care cell" is intended to mean a cell used for manufacturing / multiplying the vector. During growth or multiplication of the donor host cell, the copy number of the vector increases and at the same time cytosine in CpG motifs is replaced by a cytidine equivalent, when the cytosine equivalent is added in a sufficient amount to the growth medium used for growth of the donor host cell. The donor host cell is different from the recipient host cell, such as the donor host cell being a bacterial cell or a viral particle and the recipient host cell being an animal or plant cell.

THE VECTOR OF THE INVENTION According to the invention, the vector is a vector comprising a nucleotide sequence, in which one or two cytosines in at least one CpG motif have been replaced by a cytosine equivalent which is resistant to methylation.

By providing said vector, which is resistant to methylation, the expression of nucleotide sequences placed in the vector is maintained to a level higher than the methylated form of the vector. This maintains the function of the vector. Furthermore, the replacement of cytosine with cytosine equivalents can be used to regulate the expression of nucleotide sequences. A decrease or increase in the methylation level leads to different expression levels, so that an increased methylation level leads to a decrease in the expression level. By replacing cytosine with a cytosine equivalent, it is possible to avoid methylation problems, which can occur when the vector is introduced into a recipient host cell. In a first embodiment, cytosine is replaced with a cytosine equivalent that replaces the 5-position in the pyrimidine ring of the cytosine contained in the CpG motif.

The cytosine equivalent may be N, O or C-X. X in C-X may be a low or non-electro group, such as ethyl or methoxy. Furthermore, cytosine can be replaced by a cytidine derivative, such as 5-azacytidine and / or S-azadeoxycytidine. 5-azadeoxycytidine may also be called s-Triazin-2 (1H) -one, 4-amino-1- (2-deoxy-.beta.-D-erythropo-ran Jranosyl- (SCI), 1,3,5-Triazin- 2 (1H) -one, 4-amino-1- (2-deoxy-beta-D-erythropentofuranosyl) - (9Cl), S-aza-T-deoxycytidine or Decitabine (USAN).

The number and distribution of cytosine counterparts within the CpG motifs of the vector inhibit methylation of the 5-position in the pyrimidine ring, leading to the retention of the expression of the nucleotide sequence encoding polypeptides located in the vector. An increased number of incorporated cytosine counterparts in the nucleotide sequence region encoding a polypeptide present in the vector results in an increased ability to retain the expression of nucleotide sequences present in that region of nucleotide sequences. An increased number of incorporated cytosine counterparts in other regions of the vector may indirectly affect the maintenance of a nucleotide sequence region encoding a polypeptide. Examples of nucleotide sequence regions that indirectly affect other nucleotide sequence regions are control sequences such as promoters.

A CpG motif comprises two cytosines that are capable of being replaced by a cytosine equivalent. Either one or both are replaced. In case your cytosine equivalents are incorporated into the vector, the cytosine equivalents can be either incorporated with one and the same strand or with both strands.

The cytosine counterparts are incorporated with the vector during the copying of the vector.

The number of cytosine equivalents and their distribution leads to a resistance fl to methylation of said cytosine equivalents. The presence of said cytosine equivalents maintains the expression of nucleotide sequences comprising cytosine equivalents to an acceptable level.

In one embodiment, at least 1% (eg 1-5%) of the CpG motifs are replaced with cytosine equivalents, such as 5% (eg 5-10%), 10% (eg 10-20%), 20% (eg 20-30%), 30% (eg 30-40%), 40% (eg 40-50%), 50% (eg 50-60%). Preferably from 518,759 q is replaced from about 1 to about 100% of the cytosines with cytosine equivalents, such as from 1 to about 95% or from about 5 to about 95%. The cytosine counterparts are incorporated either into one or both strands of the nucleotide sequence of the vector.

The vector may comprise at least one gene or part of a gene. Which gene depends on what the vector is to be used for. A possible gene candidate may be one that gives rise to an immune response against indigestion and thus useful for the purpose of vaccinating humans. Furthermore, the vector may comprise one or more control sequences.

Control sequences that make it possible to maintain and multiply the vector in an appropriate donor host cell and / or control sequences that make it possible to maintain and / or multiply the vector in the recipient host cell. Additional control sequences may occur, which make it possible to express a gene and / or part of a gene in a recipient host cell to an appropriate level of expression. The choice of control sequences depends on the desired level of expression of the gene or part of the gene and one skilled in the art will be able to identify such control sequences.

The vector can be any vector, as long as the vector has the ability to multiply in values (receiver and / or donor), i.e. has a copy start. It should be understood that not all vectors and control sequences lika act equally well to express an interesting nucleotide sequence. Nor will a host work as well with the same expression system. However, one skilled in the art is able to make a selection among these vectors, control sequences and hosts without undue experimentation.

When selecting a vector, for example, values must be taken into account, since the vector must be multiplied in the donor and / or recipient host cell or be able to be incorporated with the chromosome of the recipient host cell. The copy number of the vector, the ability to control the copy number and the expression of other proteins that the vector encodes, such as selection markers, must also be considered. When choosing a control sequence, a number of factors must be taken into account. These include, for example, the relative strength of the sequence, its controllability, and its compatibility with the nucleotide sequence encoding the polypeptide of interest, particularly with respect to possible secondary structures. Hosts should be selected for their compatibility with the selected vector, the toxicity of the polypeptide encoded by the nucleotide sequence, their secretion properties, their ability to week and so on. c o u on 518 759 and u u a. ». u n a o ø. - | . ~ a polypeptide correct, their fermentatioris? or culture conditions and ease of purification of the products encoded by the nucleotide sequence.

The vector may be an autonomously replicating vector, i.e. a vector that exists as an extrachromosomal unit, where the copying is independent of the chromosomal copying, eg a plasmid. Alternatively, the vector is one which is incorporated with the recipient host cell genome and copied together with the chromosome (s) into which it has been incorporated. A vector incorporated with the genome of the recipient host cell may be advantageous to use, as a particular gene and / or part of a gene and / or control sequence needs to be protected from future possible methylation problems. Methylation can occur by copying the genome with which the vector was incorporated. Another example is when there is a need to remove a particular gene and / or part of the gene and / or control sequence present in a genome. The expression of the gene and / or part of the gene and / or the control sequence may be reduced due to the methylation and replacement of such a methylated gene and / or part of the gene and / or control sequence with an unmethylated form may possibly increase the expression of the gene relative to it. methylated. The compensation can be carried out using methods such as homologous recombination. The gene and / or part of the gene and / or the control sequence can be regulated up or down by means of such substitutions. A vector comprising one or cyt your cytidine equivalents, which is incorporated with a genome, may, upon copying the genome, give rise to new copies of the genome comprising said vector without one or cyt your cytidine analogs. However, the new copy of the genome, which contains a vector, may still be resistant to methylation, even if no cytidine analog is present in the new copy of the vector placed in the new copy of the genome.

The vector may be an expression vector in which the nucleotide sequence encoding the polypeptide of the invention is operably linked to additional segments needed for transcription of the nucleotide sequence. The vector is usually obtained from plasmid DNA or viral DNA. A number of suitable expression vectors for expression in the host cells mentioned herein are commercially available or described in the literature.

Useful expression vectors for eukaryotic hosts include, for example, vectors comprising control sequences from SV40, bovine papillomavirus, adenovirus and cytomegalovirus. 518 759 .. .. ...: .... _ '1 \ Specific vectors are, for example, pCDNA3. 1 (+) \ Hyg (Invitrogen, Carlsbad, CA, USA) and pCI-neo (Stratagene, La Jolla, CA, USA). Useful vectors for yeast cells include the Zu plasmid and derivatives thereof, the POT1 vector (US 4.93 1,373), the pJSO37 vector described in Okkels, Ann. New York Acad. Sci. 782, 202-207, 1996 and pPICZ A, B or C (Invitrogen). Useful vectors for insect cells include pVL941, pBG3l I (Cate et al., "Isolation of the Bovine and Human Genes for Mullerian Inhibiting Substance and Expression of the Human Gene in Animal Cells", Cell, 45, pp. 685-98 (1986) , pBluebac 4.5 and pMelbac (both available from Invitrogen) Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E. coli, comprising pBR322, pET3a and pET12a (both from Novagen Inc., WI, USA), plasmids for broader host selection, such as RP4, phage DNA, eg the large selection of derivatives of phage lambda, eg NM898 and other DNA phages, such as M13 and ament lamentous single-stranded DNA phages Examples of suitable viral vectors are adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, herpes vectors and cytomegaloviral vectors.

Other vectors that can be used in the invention include those that allow the nucleotide sequence encoding the polypeptide to be propagated in copy numbers. Such propagating vectors are well known in the art. They include, for example, vectors that can be amplified by DHFR amplification (see, e.g., Kaufman, U.S. Patent No. 4,470.46 1, Kaufman and Sharp, "Construction Of A Modular Dihydrafolate Reductase cDNA Gene: Analysis Of Signals Utilized For Efficient Expression"). Mol. Cell. Biol., 2, pp. 1304-19 (1982)) and glutamine synthetase ("GS") proliferation (see, for example, US 5,122,464 and EP 33 8,841).

The recombinant vector may further comprise a DNA sequence which enables the vector to be copied into the host cell in question. An example of such a sequence (when the host cell is a mammalian cell) is the SV40 copy start. When the host cell is a yeast cell, suitable sequences which enable the vector to be copied are the yeast plasmid 2u copying genes REP 1-3 and the copying start.

The vector may also include a selection marker, for example a gene whose product complements a toxin-related deficiency in the host cell, such as the gene encoding dihydrofolate reductase (DHFR) or the Schizosaccharomyces pombe TPI gene (described 518 759 k. By PR Russell, Gene 40, 1985 , pp. 125-136) or one which confers resistance to a drug, eg ampicillin, kanamycin, tetracycline, chloramphenicol, neomycin, hygromycin or methotrexate. For Saccharomyces cerevísíae, the selection markers include ura3 and leu2. For lamentous fungi, selection markers include amdS, pyrG, arcB, niaD and sC.

The term "control sequences" as defined herein includes all parts necessary or advantageous for expression of the polypeptide of the invention. Each control sequence may be natural or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader sequence, polyadenylation sequence, propeptide sequence, promoter (inducible or constitutive), enhancer or upstream activating sequence, signal peptide sequence, and transcription terminator. At least the control sequences comprise a promoter.

A large number of control sequences can be used in the present invention. Such useful control sequences include the control sequences associated with structural genes of the foregoing expression vectors as well as any sequence known to control the expression of genes in prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.

Examples of suitable control sequences for directing transcription in mammalian cells include early and late promoters of SV40 and adenovirus, eg adenovirus 2 major late promoter, MT-1 (metallothionein) promoter, cytomegalovirus between early human or rodent cytomegalovirus (CMV), the human elongation factor lot (EF-lot) promoter, the minimal heat shock Drosophila protein 70 promoter, Rous Sarcoma Virus (RSV) promoter, the human ubiquitin C (UbC) promoter, the human growth factor hormone terminator, SV40 or adenovirus Elb region Coadenylation sequencing signals (Kozak, M., J. Mol. Biol. August 20, 1987, 1996 (4): 947-50).

To enhance expression in mammalian cells, a synthetic intron can be introduced into the untranslated Si region of the nucleotide sequence encoding the polypeptide. An example of a synthetic intron is the synthetic intron from plasmid pCI-Neo or 518,759 few from the β-globin gene (obtained from Promega Corporation, WI, USA). In addition, an IRES component may be included.

Examples of suitable control sequences for directing transcription in insect cells include the polyhedrin promoter, the P1O promoter, the Autographa calzfornica polyhedrosvirus basic protein promoter, the baculovirus intermediate gene 1 promoter and the baculovirus 39K delayed gene promoter and the SV40 polyadenyl sequence. Examples of suitable control sequences for use in yeast host cells include the promoters of the yeast excitatory system, the yeast triosphosphatase isomerase (TPI) promoter, promoters of yeast glycolytic genes or alcohol dehydrogenase genes, the ADH2-4c promoter and the inducible GAL promoter. Examples of suitable control sequences for use in lamentous fungal host cells include the ADH3 promoter and terminator, a promoter derived from the genes encoding Aspergillus oryzae TAKA amylastriosphosphate isomerase or alkaline protease, an A. niger ot-amylase, A. nulagan or A. nidulans acetamidase, Rhizomucor meme aspartic acid proteinase or lipase, TPI 1 terminator and ADH3 terminator. Examples of suitable control sequences for use in bacterial host cells include promoters for the lac system, the trp system, the TAC or T RC system and the major promoter regions of phage lambda.

The presence or absence of a signal peptide depends, for example, on the expression host cell used to produce the polypeptide to be expressed (whether it is an intracellular or extracellular polypeptide) and whether it is desirable to obtain secretion. For use in lamentous fungi, the signal peptide may conveniently be obtained from a gene encoding an Aspergillus sp. amylase or glucoamylase, a gene encoding a Rhízomucor míeheí lipase or protease or a Humícola lanugínosa lipase. The signal peptide is preferably obtained from a gene encoding A. oryzae TAKA amylase, A. niger neutral ot-amylase, A. niger acid-stable amylase or A. niger glucoamylase. For use in insect cells, the signal peptide may conveniently be obtained from an insect gene (see also WO 90/05783) such as the Lepidopteran manduca sixth adipokinetic hormone precursor (see also US 5,023,328), honey bi melittin (Invitrogen), ecdysteroid UDP-glucosyltrans ( et al., Protein Expression and Purification 4, 349-357 (1993) or human pancreatic lipase (hpl) 518 759 \ '- \ (Methods in Enzymology, 284, pp. 262-272, 1997). A preferred signal peptide for uu for use in mammalian cells is the signal peptide from hG-CSF or the murine Ig kappa light chain (Coloma, M. (1992) J. Imm. Methods 152289-104). For use in yeast cells, suitable signal peptides have been found to be ot factor signal peptide from S. cereviciae (see also US 4,870,008), a modified carboxypeptidase signal peptide (see also LA Valls et al., Cell 48, 1987, pp. 887-897), yeast BARI signal peptide (see also WO 87/02670 ), yeast aspartic acid protease 3- (YAP3) signal peptide (see below) re M. Egel-Mitani et al., Yeast 6, 1990, pp. 127-137) and the synthetic leader sequence TA57 (WO 98/32867).

Any suitable donor host cell can be used to maintain and manufacture the vector of the invention, such as a eukaryotic or prokaryotic cell, eg bacteria, fungi (including yeast), plants, insects, mammals or other suitable animal cells or cell lines as well as transgenic animals or plants. . The donor host cell may be a donor host cell belonging to a GMP (Good Manufacturing Practice) certified cell line, such as a mammalian cell line. Examples of bacterial donor host cells include gram-positive bacteria such as strains of Bacillus, eg B. brevis or B. subtílis, Pseudomonas or Streptomyces or gram-negative bacteria such as strains of E. coli. Examples of suitable ament lamentous fungal donor host cells include strains of Aspergillus, eg A. oryzae, A. niger or A. nidulans, F usarium or T richoderma.

Examples of suitable yeast donor host cells include strains of Saccharomyces, eg S. cerevísíae, Schizosaccharomyces, Klyveromyces, Pichía, such as P. pastoris or P. methanolíca, Hansenula such as H Polymorpha or Yarrowía. Examples of suitable insect donor host cells include a Lepidoptora cell line such as Spodoptera frugíperda (Sf9 or Sf21) or T richoplusioa ní cells (High Five) (US 5,077,214). Examples of suitable mammalian donor host cells include Chinese hamster ovary (CHO) cell lines (eg CHO-K1, ATCC CCL-61), green monkey cell lines (COS) (eg COS 1 (ATCC CRL-1650), COS 7 (ATCC CRL-1651) )), mouse cells (eg NS / O, kidney (BHK) hamster cell lines (eg ATCC CRL-1652 or ATCC CCL-10) and human cells (eg HEK 293 (ATCC CRL-1573)), as well as plant cells Additional suitable donor host cell lines are known in the art and are obtained from public repositories such as the American Type Culture Collection, Rockville, Maryland.518 759 In a particular embodiment, the invention relates to a nucleotide sequence which IIII... '. -... -. n was obtained from a donor host containing the nucleotide sequence as part of the vector.

The vector and transducer values are a vector and a transducer host as described above.

The nucleotide sequence may be a gene and / or part of a gene and / or one or more of the control sequences as mentioned above. An example of gene or genes of interest are genes encoding cell nuclear protein, such as genes encoding the intracellular nucleus protein that can be used for vaccination. Another example is the gene encoding tyrosine hydroxylase, which may be useful in the treatment of Parkinson's disease. A third example is genes encoding factor X or VIII involved in the blood coagulation complex or interferon gamma. In addition, all types of genes that can be used in gene therapy such as gene candidates that can be used in the treatment of cancer are suitable for use according to the invention, such as thymidine kinase from Herpes Simplex Virus (HSV).

In another embodiment, the invention relates to a polypeptide produced by the donor host cell, wherein the polypeptide is encoded by a nucleotide sequence that is part of the vector according to the invention and is described above.

In another embodiment, the invention relates to a pharmaceutical composition comprising the above-described vector and / or donor host cell and / or a nucleotide sequence obtained from the vector and / or a polypeptide and a pharmaceutically acceptable diluent, carrier, adjuvant or excipient.

The vector, nucleotide sequence, polypeptide or pharmaceutical composition as described above can be transferred into a recipient host cell using a suitable transformation method such as electroporation, microprojectile firing or liposome-mediated delivery.

The vector, nucleotide sequence, polypeptide or pharmaceutical composition as described above may be used in treatment and / or in diagnostics.

The vector, nucleotide sequence, polypeptide or pharmaceutical composition as described above may be used for the manufacture of a medicament for use in treatment and / or diagnostics. 518 759 Ho.

METHOD OF MANUFACTURING THE VECTOR ACCORDING TO THE INVENTION An embodiment according to the invention relates to a method which reduces the methylation of CpG motifs in a vector in a recipient host, the method comprising replacing at least one cytosine in a CpG motif with a cytosine equivalent, the vector has described above.

According to another embodiment of the invention, the method comprises the steps of: providing a vector, transferring the vector into a donor host cell, culturing the donor host cell containing the vector in a growth medium consisting of an appropriate amount of cytosine equivalents and harvesting the propagated vector comprising inserted cytosine equivalents in one or two cytosine equivalents at least one CpG motif.

The vector is a vector as described above and the donor host is a donor host cell as described above.

The vector and donor values are selected depending on the purpose of creating the specific vector with inserted cytosine equivalents and a person skilled in the art can perform this selection. The vector is entered into sensor values using a suitable method, which depends on which sensor value has been selected. The introduction of a vector into a bacterial donor host cell can be accomplished, for example, by protoplast transformation (see, e.g., Chang and Cohen, 1979, Molecular General Genetics 1682111-115) using competent cells (see, e.g., Young and Spizizin, 1961, Journal). of Bacteriology 822823-829 or Dubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56: 209-221), electroporation (see e.g. Shigekawa and Dower, 1988, Biotechniques 6: 742-751) or conjugation (see e.g. Koehler and Thome, 1987, Journal of Bacteriology 169: 5771-1 5278). Fungal cells can be transformed using protoplast formulation. Transformation of protoplasts and regeneration of the cell wall takes place in a manner known per se. Suitable methods for transforming Aspergillus host cells are described in EP 238 023 and US 5,679,543. Suitable methods for transforming F usarium species are described by Malardier et al., 1989, Gene 78: 147-156 and WO 96/00787. Yeast can be transformed using methods described by Becker and Guarente, I Abelson, J .N. and Simon, M.I., eds., Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp. 182-187, Academic Press, Inc., New York, Ito et al., 518, 759 t? 1983, Journal of Bacteriology 1532163, Hinnen et al., 1978, Proceedings of the National Academy of Sciences USA 75: 1920 and as described by Clontech O! Laboratories, Inc., Palo Alto, CA, USA (in the YeastmakerTM Yeast Transformation System Kit Product Protocol). Transformation of insect cells and production of heterologous polypeptides can be performed as described by Invitrogen. Methods for introducing exogenous DNA into mammalian donor host cells include calcium phosphate-mediated transfection, electroporation, DEAE-dextran-mediated transfection, liposome-mediated transfection, viral vectors, and the transfection method described by Life Technologies Ltd, Paisley, U.K.. for example, in Ausbel et al. (ed.), 1996, Current Protocols in Molecular Biology, John Wiley & Sons, New York, USA.

In the production methods of the present invention, the cells are grown in a growth medium suitable for maintaining and / or producing the vector using methods known in the art. For example, the cells can be grown by shaking fl ash culture, small scale or large scale fermentation (including continuous, batch, fedbatch or solid phase fermentations) in laboratory or industrial fermenters, where the fermentation is carried out in a suitable medium and under conditions that allow the vector, nucleotide sequence or polypeptide to be expressed and / or isolated. The vector, nucleotide sequence or polypeptide can be used in the chemical or pharmaceutical industry. The cultivation takes place in a suitable growth medium comprising carbon and nitrogen sources and inorganic salts using methods known in the art.

Suitable media are obtained from commercial suppliers or can be produced according to published compositions (eg in catalogs from the American Type Culture Collection). The culture of mammalian donor cells is performed according to established methods, for example as described in (Animal Cell Biotechnology, Methods and Protocols, edited by Nigel Jenkins, 1999, Human Press Inc., Totowa, New Jersey, USA and Harrison MA and Rae IF, General Techniques of Cell Culture, Cambridge University Press 1997).

The cytosine equivalent described above is added to the growth medium in an appropriate amount sufficient to obtain a vector with a sufficient amount of cytosine equivalents incorporated with the nucleotide sequence to obtain a desired expression of the nucleotide sequences encoding the polypeptides. vu. nao: cytosine equivalents depend on fl your factors including the vector size, the amount of CpG motifs and the host cell In eukaryotic cells, for example, 10 μM 5-azacytidine and / or 5-azadeoxycytidine are often used.

After culture, donor values are harvested by, for example, centrifugation and the vector is recovered by methods known in the art. Examples of methods are those mentioned in Maniatis et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press) (1989) and Qiagen Inc.

The amount and distribution of cytosine equivalents present in the vector after preparation in a suitable donor host can be determined using, for example, labeled 5-azacytidine and / or S-azadeoxycytidine, HPLC, GC or TC.

KIT ACCORDING TO THE INVENTION Accordingly, the invention relates to a kit comprising a vector as described above and / or a donor host cell as described above comprising a description of how the vector and / or vector and the donor host cell are used.

This kit may further comprise a growth medium suitable for the ability of the donor host cell to grow and multiply the vector.

This kit can be used for the production of a polypeptide in industry or in a method of treating a mammal suffering from a disorder or in need of vaccination. Examples of disorders are disagreement and cancer.

PHARMACEUTICAL USE AND FORMULATIONS According to one embodiment of the invention, the vector, donor values, nucleotide sequence, polypeptide or pharmaceutical composition of the invention may be used for your applications comprising vaccination and gene therapy.

However, when the vector and / or donor values are used to treat a disorder or vaccination, it is important that the nucleotide sequences that create the vector are nucleotide sequences (eg, copy initiation and control sequences) that retain their activity as expression of a polypeptide in recipient values. , i.e. patient neo- uu. c u o n u a a v a v | ø now cells. For example, in the case where the vector is used to vaccinate a human, it is important that a promoter be used upstream of the nucleotide sequence encoding a polypeptide that elicits an immune response, i.e. the promoter is active in the human cell.

The promoter may be inducible and / or continuous. In the case where an inducible promoter is used, it may be inducible by an agent which is either administered together with or after the vector and / or donor values. The promoter can be further induced by an agent produced in the patient, either locally or systemically.

For example, if plasmids encoding insulin are introduced intramuscularly by electroporation, the plasmid expresses insulin for a time and then the expression is attenuated. A methylation resistant plasmid, on the other hand, is likely to have a constant expression for a long time. If such a plasmid has a tetracycline-inducible promoter, insulin production could be controlled by oral administration of tetracycline. This type of gene therapy could be applied to patients with insulin deficiency diabetes or to hemolytic patients where a gene to express a coagulation factor is missing.

In a second embodiment, the vector / donor host, nucleotide sequence, polypeptide or pharmaceutical composition of the invention is used in the manufacture of a medicament for the treatment of disorders or vaccination, such as cancer. Today's cancer treatment focuses on other treatments than classical radiotherapy and chemotherapy. Two promising areas for cancer are antiangiogenic treatment and immunotherapy. Both treatments can utilize gene therapy to produce antiangiogenic agents or to produce immunostimulatory or toxic agents. These types of treatments can be optimized by using methylation-resistant vectors, both as pure plasmids or as viral vectors made resistant to methylation. A genetically modified adenovirus, which expresses the gene for Herpes Simplex Thymidine kinase (HSV-TK), can be produced, for example, in the presence of a described cytosine equivalent.

DNA of the adenovirus will contain this equivalent and in an infection, the adenovirus expresses higher levels of HSV-TK over a long period of time compared to an adenovirus that does not have methylation resistant DNA. Tumor cells can - c - c; | n n. n u ø v n Q ou then killed with a herpes virus drug. The killing is related to the production of HSV-TK, i.e. the higher the concentration of HSV-TK the more sensitive the tumor cell will be to the drug.

In another embodiment, the vector / donor host, nucleotide sequence, polypeptide or pharmaceutical composition of the invention is used in a method of treating a mammal which has a disorder or is in need of vaccination.

Vaccinations have traditionally been carried out using mutated or killed viruses.

Vaccination with DNA uses proteins encoded by the pathogen's DNA. Such a gene or genes is cloned into a plasmid or another vector. Plasmids encoding one or more pathogenic proteins can be injected, eg intramuscularly. The plasmid expresses the pathogenic protein or proteins recognized by the immune system of the individual.

This type of immunization then protects against subsequent infection of the pathogen.

The strength of the immune response depends on the production of the pathogenic protein. Excessive production of the pathogenic protein or proteins may not be recognized by the immune system. Consequently, immunization of both humans and animals with methylation-resistant vectors will improve efficiency.

Pharmaceutical formulations of the vector / donor values, nucleotide sequence, polypeptide of the invention are usually administered in a composition comprising one or more pharmaceutically acceptable carriers or excipients. Such pharmaceutical compositions can be prepared in a manner known in the art, are sufficiently stable in storage and suitable for administration to humans and animals. The pharmaceutical composition may be lyolized.

"Pharmaceutically acceptable" means a carrier or excipient which, when used and the concentrations used, does not cause any adverse effects in the patient to whom it is administered. Such pharmaceutically acceptable carriers or excipients are well known in the art (see Remington °'s Pharmaceutical Sciences, 18th Edition, AR Gennaro, ed. Mack Publishing Company (1990) and Handbook of Pharmaceutical Excipients, 3rd Edition, A. Kibbe, ed., Pharmaceutical Press (2000). 518 759 3- \ The pharmaceutical composition can be mixed with adjuvants such as lactose, un ~ «ø ~ q ø u - | ~ I oou nu sucrose, starch powder, cellulose esters of alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, acacia, gelatin, sodium alginate, polyvinylpyrrolidone and / or polyvinyl alcohol and are tableted or encapsulated for conventional administration.

Alternatively, they can be dissolved in saline, water, polyethylene glycol, propylene glycol, ethanol, oils (such as corn oil, peanut oil, cottonseed oil or sesame oil a), gum tragacanth and / or various buffers. Other adjuvants and modes of administration are well known in the pharmaceutical art. The carrier or diluent may comprise time delay materials, such as glyceryl monostearate or glyceryl distearate, alone or with a wax, or other materials well known in the art.

The pharmaceutical compositions may be subjected to conventional pharmaceutical methods such as sterilization and / or contain customary adjuvants such as preservatives, stabilizers, wetting agents, emulsifying agents, buffers, fillers, etc., which are described elsewhere herein.

The pharmaceutical composition according to the invention can be administered locally or systemically as on top, intravenously, orally, parenterally or as an implant and also rectal use is possible. Suitable solid or liquid pharmaceutical formulations are, for example, granules, powders, tablets, coated tablets, (micro) capsules, suppositories, syrups, emulsions, suspensions, creams, sprays, drops or injectable solution in ampoule forms and also preparations with prolonged release of active compounds. , in which the excipients, diluents, adjuvants or carriers of the preparations are used as they are usually made and as described above.

The pharmaceutical composition is administered to a patient in a pharmaceutically effective dose. By "pharmaceutically effective dose" is meant a dose sufficient to produce the desired effects relative to the condition for which it is administered. The exact dose depends on the activity of the compound, the route of administration, the type and severity of the disorder. Depending on the patient's age and body weight, different doses may be needed. The administration of the dose can be performed both by a simple administration in the form of a single dose unit or also your smaller dose units and 518 759: Also read through your administrations of divided units at specific intervals. To -. | - ø c u u u ~ n - Q - o: example vaccination.

The pharmaceutical composition of the invention may be administered alone or in combination with other therapeutic agents. These agents may be involved as part of the same pharmaceutical composition or administered separately.

The "patient" according to the invention includes both humans and other mammals.

Consequently, the methods are applicable to both human treatment and veterinary applications.

The following examples are intended to illustrate, but not to limit the invention in any way, in any form or form, either express or implied.

EXAMPLES Example 1 Preparation of Methyl Resistant Plasmid DNA in Bacteria 1. Inoculate 2 ml of common LB medium and 50 μg / ml ampicillin in a 10 ml test tube with an E. coli containing an interesting plasmid. 2. The culture is incubated in a shaker (200 rpm) at 37 ° C overnight. The supernatant culture is then used to inoculate a 500 ml LB medium into a 2 liter culture flask. The flask is incubated at 37 ° C in a shaker until the growth of bacteria reaches OD (650) of 0.5 to 0.8. 4. Sideoxiazacytidine or other deoxycytidine analog is added to a concentration of 10 μM and chloramphenicol is added to a final concentration of 180 μg / ml. 5. The culture is incubated at 37 ° C on a shaker as above overnight. The bacteria are pelleted by conventional centrifugation and plasmid DNA is isolated according to Qiagen Inc. Maxiprep standard protocol.

Example 2 Preparation of adenovirus with methylation resistant DNA 518 7.59 Qjš n | o u nu. Spread 911 or 293 cells in standard T-75 flasks to be 60-90% conducive at the time of infection (approximately 1x107 cells / T-75). Generally, 15 to 20 T-75 pistons are sufficient to make a high titer stock.

Infect cells with virus supernatant with an infection multiplication (MOI) of 5 to 10 PFU (plaque-forming units) per cell.

. On day 2, 5'-deoxiazacytidine is added to a concentration of 10 μM.

. When all the cells have grown together and about half of the cells have detached (usually at 3 to 4 days after infection), harvest and merge all the flasks. Centrifuge for 5 minutes in a bench centrifuge (~ 500 g) and remove the supernatant.

. Slurry the pellet in 8.0 ml of sterile PBS. Perform 4 cycles of freezing / thawing / vortex. Centrifuge the lysate in the Sorvall HS4 rotor at 6000 rpm (7000 g) at 4 ° C for 5 minutes.

. Weigh 4.4 g of CsCl into a 50 ml conical tube, transfer 8.0 ml of clear virus supernatant to the tube and mix well by vortexing. Transfer the CsCl solution (ca. 10 ml, density 1.35 g / ml) to a 12 ml polyallomer tube for a SW41 rotor. Cover the tube with 2 ml of mineral oil. Prepare a balance tube. Centrifuge the gradient in the SW 41 rotor at 32,000 rpm, 10 ° C, for 18 to 24 hours.

. Collect the virus fraction (about 0.5 to 1.0 ml) with a 3 ml syringe and an 18g needle.

Insert the needle from below through the tube from below. Mix with equal volume 2X Storage Buffer (2X Storage Buffer = 10 mM Tris, pH 8.0, 100 mM NaCl, 0.1% BSA and 50% glycerol, sterilized). Store the virus stock at -20 ° C.

. Check for virus titer with GFP (preferred) or with a plaque assay (see below) or immunohistochemical staining or simply read OD at 260 nm. To read OD, add 15 μl virus to 15 μl blank solution (blank solution = 1.35 g / ml CsCl mixed with equal volume 2X Storage Buffer) plus 100 μl TE / 0.1% SDS, vortex for 30 seconds, centrifuge for 5 minutes . Measure A260. An A260 unit contains ~ l x 1012 virus particles (particle infectious particles should be around 2011). 518 759 SLM o o e v o ø. v c u u Q ø c | | Example 3 Measurement of the degree of incorporation of S-azadeoxycytidine into DNA.

Method 1 T-deoxiazacytidine is added to the media containing growing cells to a concentration of 10 pM. Also add a trace amount of MC-labeled deoxiazacytidine, eg 5 pCi. If the specific activity of labeled azacytidine is 500 Ci / mmol, this is equal to 10 pmol azacytidine. If the volume used is 100 ml, the concentration of the radiolabeled nucleotide is 100 pM. The ratio of the cold to labeled nucleotide is then 100,000: 1. The radioactivity of the isolated DNA is measured in a scintillation counter and the incorporation is estimated depending on the efficiency of the target (1 ci = 1 x 10 ”tip).

Method 2 Add 5'-deoxiazacytidine to the media containing growing cells to a concentration of 10 pM. The azacytidine molecule can be separated from cytidine by HPLC. The isolated DNA is treated with DNAase I, so that a high concentration of free nucleotides is formed. The samples are injected on a suitable HPLC column and the nucleotides are separated into cytidine, thymidine, guanine, adenine and 5'-azacytidine, respectively.

The concentration of azacytidine is measured in relation to cytidine.

Claims (35)

o Q | u u nu 5 1 8 7 5 9 aš PATENTKRAV A vector comprising a nucleotide sequence, wherein one or two cytosines in at least one CpG motif have been replaced with a cytosine equivalent that is resistant to methylation. The vector of claim 1, wherein the cytosine equivalent replaces the S position in the pyrimidine ring of the cytosine present in the CpG motif with N, O or C-X. The vector of claim 2, wherein the cytosine equivalent is a cytidine derivative. The vector of claim 3, wherein the cytidine derivative is S-azacytidine and / or 5-azadeoxycytidine. The vector of claim 2, wherein X is a low or non-electrical group. The vector of claim 5, wherein the low non-electrophilic group is selected from the group consisting of ethyl and methoxy. The vector of any one of the preceding claims, wherein the replaced cytosine (s) are located in one and the same DNA strand or in both DNA strands of the vector. The vector of any one of the preceding claims, wherein the vector is selected from the group consisting of plasmids, bacteriophages, phagemids and viral vectors. The vector according to any one of the preceding claims, wherein the number of cytosine equivalents and the distribution of cytosine equivalents alter the nucleotide sequences comprising said cytosine equivalents so that they become methylation resistant and retain the expression of the nucleotide sequences comprising CpG motifs containing cytosine equivalents. 10. lO. The vector of any one of the preceding claims, wherein at least 1% of the CpG motifs have been replaced with cytosine equivalents. 11. ll. The vector of claim 10, wherein at least 5% of the CpG motifs have been replaced with cytosine equivalents. The vector of claim 11, wherein at least 10% of the CpG motifs have been replaced with cytosine equivalents. The vector of claim 12, wherein at least from about 1% to about 100% of the CpG motifs have been replaced with cytosine equivalents. 518 759: Le u - ~ n | o u ~ u u u v u n oo The vector of any one of the preceding claims, wherein the vector comprises at least one gene or part of a gene. The vector according to any one of the preceding claims, wherein the vector comprises one or fl your expression control sequences. A donor host cell comprising a vector according to any one of claims 1-15. The donor host cell of claim 16, wherein the donor cell is a eukaryotic or a prokaryotic cell. The donor host cell of claim 17, wherein the donor host cell is selected from the group consisting of isolated bacteria, fungi, plants, insects, mammals or other suitable animal cells or cell lines as well as transgenic animals or plants. The donor host cell according to any one of claims 15-18, wherein the donor host cell is a host cell belonging to a GMP certified cell line. The donor host cell of claim 19, wherein the cell line is a mammalian cell line. A nucleotide sequence obtained from the donor host cell according to any one of claims 16-20, the nucleotide sequence is part of the vector according to any one of claims 1 to 15. The nucleotide sequence of claim 21, wherein the nucleotide sequence is one or fl your genes and / or part of one or fl your genes and / or one or fl your expression control sequences. A polypeptide made by the donor host cell of any one of claims 16-20, the nucleotide sequence encoding the polypeptide is part of the vector of any one of claims 1-15. A pharmaceutical composition comprising a vector according to any one of claims 1-15 and / or the donor host cell according to any one of claims 16-20 and / or the nucleotide sequence according to claims 21-22 and / or the polypeptide according to claim 23 and a pharmaceutically acceptable diluent, carrier, adjuvant or excipient. A method of reducing the methylation of CpG motifs in a vector in a recipient host, the method comprising replacing at least one cytosine in a CpG motif with a cytosine equivalent according to any one of claims 1-15. The method of claim 25, wherein the method comprises the steps of i) providing a vector, ii) transferring the vector into a donor host cell, iii) culturing the donor host cell containing the vector in a growth medium consisting of an appropriate amount of 518,759 α-cytosine equivalents and iv) harvesting the amplified vector comprising uu - oa »~ nnuonn ~ - - uv incorporated cytosine counterparts in at least one CpG motif. The method of claim 26, wherein the cytosine equivalent is a cytidine derivative. The method of claim 27, wherein the cytidine derivative is 5-azaeytidine and / or 5-azadeoxycytidine. The method of any one of claims 26-28, wherein the harvested vector is a vector according to any one of claims 1-15. The method of any of claims 25-29, wherein the donor host cell is a donor host cell according to any of claims 16-20. The method according to any one of claims 25-29, which is used in small or large scale production of a nucleotide sequence and / or a polypeptide in the chemical industry such as the pharmaceutical industry. A kit comprising a vector according to any one of claims 1 to 15 and / or a donor host cell according to any one of claims 16 to 20. A method of transferring a vector according to any one of claims 1-15, a nucleotide sequence according to any one of claims 21-22, a polypeptide according to claim 23 or a pharmaceutical composition according to claim 24 into a recipient host cell, the method being selected from the list consisting of electroporation, microprojectile shelling and liposome-derived delivery. A vector according to any one of claims 1-15, a donor host cell according to claims 16-20, a nucleotide sequence according to any one of claims 21-22, a polypeptide according to claim 23 or a pharmaceutical composition according to claim 24 for use in treatment and / or in diagnostics. Use of a vector according to any one of claims 1-15, a donor host cell according to claims 16-20, a nucleotide sequence according to any one of claims 21-22, a polypeptide according to claim 23 or a pharmaceutical composition according to claim 24 for the manufacture of a medicament for use in treatment and / or in diagnostics.