WO2002102417A1 - Vector and the use thereof in gene therapy methods - Google Patents

Abstract

The invention relates to a vector, which is particularly suitable for somatic gene therapy, to pharmaceutical compositions containing the vector and to the use of said vector. The inventive vector contains at least one nucleotide sequence that codes for a translocation sequence, selected from the group composed of nucleotide sequences that code for the protein HSV VP22, an HIV-Tat translocation domain and the antennapedia peptide.

Description

 Vector and its use in gene therapy processes

description

The present invention relates to a vector, in particular a plasmid vector for gene therapy purposes, in particular for somatic gene therapy, pharmaceutical compositions containing this plasmid vector and uses and methods using this plasmid vector.

Gene therapy methods are methods for correcting hereditary diseases using genetic engineering methodology by replacing one or more defective genes, by additionally introducing the normal gene or by introducing a gene which compensates for the effect of the defective gene. Gene therapy methods are currently still in the experimental stage on animals. A distinction is made between germline gene therapy and somatic gene therapy. In the course of germline gene therapy, the genetic defect in the early embryonic stage is remedied, so that the germ cells of the individual are also affected and the changed genetic information is passed on to the offspring. Somatic gene therapy, on the other hand, eliminates genetic defects solely in organisms developed in the body cells, for example by transmitting a gene that has normal function. The genetic material passed on to the offspring therefore still contains the genetic defect. Usually, in the context of somatic gene therapy, the body cells to be treated are first removed from the body, finally, the desired genetic changes in the cells are carried out in vitro, and as a rule selected cells transformed by transforming the target cells with the gene used for gene therapy and then reimplanted in the patient.

Knowledge of the molecular causes of the defect to be treated is a prerequisite for any successful gene therapy. A prerequisite is also the provision of the largest possible number of cells transformed with the desired gene. The transformation, that is to say the introduction of genetic material into the cells, usually takes place by means of chemical-physical methods, for example microinjection of DNA, or by using vectors, for example retroviruses, adenoviruses, herpes simplex viruses, DNA-containing liposomes or plasmid vectors.

For example, from WO 99/66061, WO 99/04026, WO 96/37623 or WO 96/14332, viral vectors are known which are proposed for use in gene therapy processes and are capable of generating genetics Introduce material into target cells.

Polymeric gene carriers are known from WO 99/59546 which can introduce nucleic acids into cells. WO 98/40502 describes peptide- or protein-containing compositions which, in addition to a transfection agent, also contain nucleic acids and can be used for gene therapy processes. US 5,804,604 and US 5,674,980 describe the use of a plasmid for therapeutic purposes. This plasmid encodes a fusion protein, comprising a basic region of the HIV-TAT transport protein (HIV: human immunodeficiency virus) and the Papillo avirus E2 repressor. The fusion protein formed from the TAT transport protein part and the E2 repressor protein serves as a transport vehicle for the introduction of peptides, macromolecules or smaller molecules such as nucleic acids or polysaccharides into target cells.

WO 87/02989 describes the HIV TAT-3 protein and plasmids which have nucleic acid sequences coding for this protein. It is disclosed that the recombinant TAT-3 protein can be used for the diagnosis and therapy of HIV infections.

Finally, Schwarze et al. (Science

(1999) 285, 1569-1572 the intraperitoneal injection of a fusion protein comprising the HIV-TAT protein into mouse cells.

The transformation processes described in the context of gene therapy have the disadvantage of comparatively low efficiency. The number of transformed cells necessary for a gene therapy promising approach is difficult or impossible to achieve. The described methods also frequently have the disadvantage that the expression rate in the transformed cells is not high enough to be expected to have a therapeutic effect. It has proven to be particularly disadvantageous that some tissues or organs such as the brain are difficult to reach for metabolic products and, because of the blood / brain barrier, cannot be supplied with proteins by other organs. Somatic gene therapy is particularly problematic in such tissues or organs.

The present invention is therefore based on the technical problem of providing means and methods which ensure the high transformation and expression rate of genes of interest which is necessary for a successful gene therapeutic method.

The present invention solves the technical problem on which it is based by providing a vector for gene therapy purposes, comprising at least one nucleotide sequence coding for a translocation sequence selected from the group consisting of a nucleotide sequence coding for the HSV VP22 protein (HSV-VP22) : Elliott and O'Hare Cell (1997) 88, 223-233), a nucleotide sequence encoding the HIV-TAT protein transduction domain and a nucleotide sequence encoding the Antennipedia peptide (Antennipedia: Derozzi et al., J. Biol. Chem. (1994) 269, 10444-10450, Helix der Ho eodomäne: RQIKIWFGNRRMKWKK), SEQ ID No. 4).

In particular, such a vector is designed as a plasmid vector, viral vector or liposome.

The present invention solves the underlying technical problem in particular by A plasmid vector comprising operatively linked to one another in the 5 'to 3' direction at least one constitutively expressing promoter, the nucleotide sequence coding for at least one translocation sequence, a NotI cloning site for the insertion of at least one nucleotide sequence which is useful in gene therapy and a polyadenylation site. According to the invention, a plasmid vector is preferably provided for gene therapy purposes, the translocation sequence comprising the 11 amino acids YGRKKRRQRRR (SEQ ID No. 2) of the naturally occurring HIV-TAT lead peptide. In one variant, the translocation sequence comprises the 11 amino acids YARAAARQARA (SEQ ID No. 5) of an HIV-TAT lead peptide modified by targeted amino acid exchanges.

According to the invention, a plasmid vector for gene therapy purposes is preferably provided, which is referred to as pCURE (FIG. 1), comprising in operative linkage and in 5 'to 3' orientation at least one constitutively expressing promoter, at least one coding for a ribosome binding site (RBS) Nucleotide sequence, at least one nucleotide sequence encoding the HIV-TAT lead peptide, in particular encoding the 11 amino acids YGRKKRRQRRR (SEQ ID No. 2), an NotI cloning site for the insertion of a nucleotide sequence which is useful for gene therapy, in particular a gene therapy-meaningful gene site, and a polyaden ,

The plasmid vectors according to the invention are characterized by a translocation sequence, in particular a nucleotide sequence coding for HIV-TAT leader peptide sequence, which is linked directly and seamlessly in reading frame to a nucleotide sequence coding for example a therapeutically meaningful protein or protein fragment, so that in the course of the transcription and translation in particular a HIV-TAT leader peptide sequence or a modified HIV Fusion protein having a TAT lead peptide sequence is formed in the target cell, that is to say the cell to be transformed or transformed. The use of a translocation leader sequence (leader peptide sequence or translocation signal) of 11 amino acids, i.e. the protein transduction domain of the HIV-TAT protein, which is preferably provided according to the invention, surprisingly enables the fusion protein formed in a target cell to be exported from the transformed cell and imported into at least one another cell, especially crossing the blood-brain barrier. It was surprisingly found that the translocation property of a modified HIV-TAT protein transduction domain, in particular with an amino acid sequence as shown in SEQ ID No. 5, as a leading peptide improves even further compared to the naturally occurring protein-transduction domain of the HIV-TAT protein is.

The fusion protein encoded by the vector according to the invention is therefore suitable for intercellular transport. The use of the plasmid vector according to the invention also leads to an increased number of transformed cells with intact protein, each with DNA-transformed cell, according to the invention. The plasmid vector according to the invention preferably also carries a translation initiation sequence linked to the leader sequence, namely a nucleotide sequence coding the RBS sequence (ribosome binding site, "Kozak sequence") and in particular all other elements required for transcription, such as a constitutively expressing promoter Example of the CMV promoter or the human beta-actin promoter for ubiquitous expression, a polyadenylation site, preferably the polyadenylation site of SV40 (= SV40pA) and at least one termination signal.

In a particularly preferred embodiment, according to which the vector without inserted foreign DNA has a size of only 2.8 kbp (FIG. 2) to 3.4 kbp (FIG. 1), the plasmid vector according to the invention is extremely small and can therefore also be large and / or record several genes or gene segments that are useful in gene therapy. The insertion of the genes or gene segments which are meaningful in gene therapy is carried out by means of the inventively preferred, that is to say only once in the vector, that is to say “unique” NotI interface, which lies immediately behind the nucleotide sequence coding for the leading peptide or overlaps with it.

According to the invention, a preferred embodiment provides that the plasmid is provided with developmental or tissue-specific or regulatable regulatory elements, for example promoters or enhancers, which are a multiple cloning site are inserted into the vector.

According to the invention, in a particularly preferred embodiment, it is provided to provide a multiple cloning site for the insertion of at least one regulatory element in the plasmid vector according to the invention, in particular this - in the 5 'to 3' direction - between a constitutively expressing promoter and a ribosome binding site insert le. In a preferred variant, the constitutively expressing is the CMV promoter.

In a particularly preferred embodiment of the plasmid vector according to the invention, the translocation sequence comprises a modified amino acid sequence which results from the amino acid sequence SEQ ID No. 2 by addition, deletion or exchange of at least one amino acid, preferably β amino acids. In particular, the amino acids glycine, lysine and / or arginine are replaced by the amino acid alanine.

According to the invention, the plasmid vector is preferred, wherein the translocation sequence comprises a modified amino acid sequence which results from the amino acid sequence SEQ ID No. 5 by addition, deletion or exchange of at least one amino acid, preferably from one to five amino acids.

According to the invention, the plasmid vector preferably has a nucleotide sequence functioning as an “N-linker”, which contains the nucleotide sequence coding for a translocation sequence. In one variant In this preferred embodiment, the nucleotide sequence functioning as an “N-linker” comprises the nucleotide sequence according to SEQ ID No. 12. In an alternative preferred embodiment, the plasmid vector according to the invention contains a nucleotide sequence which functions as a “C-linker”, in which the nucleotide sequence coding for the translocation sequence contains is. In a preferred variant of this embodiment, the nucleotide sequence functioning as a "C-linker" comprises the nucleotide sequence SEQ ID No. 13. The translocation sequence is preferably always fused to the N-terminus of a nucleotide sequence that is useful in gene therapy. However, it is conceivable that certain gene therapy makes sense - Full nucleotide sequences such as enzymes must retain the native N-terminus, either because they are only involved in the enzymatic activity in this unfused conformation or because they themselves carry specific transport signals, for example for import into the cell nucleus or into the mitochondia or for export to the extracellular matrix, in which case a cleavage of the leading peptide sequences after passage through the corresponding membrane is possible or even necessary. On the other hand, some nucleotide sequences which are useful in gene therapy, such as enzymes, have a functional C-terminus which is used to maintain d the catalytic properties must not be blocked. In order to take these two possibilities into account, the N-linkers and C-linkers according to the invention described above were constructed as an optional component of the plasmid vector according to the invention. For encoding proteins with either N- or C-terminal protein transduction domains for cloning Domain for cloning appropriate nucleotide sequences which are useful in gene therapy into the Not I interface of the plasmid vector according to the invention. According to the invention, two versions of therapy plasmids are therefore preferably provided. On the one hand pCURE2C, on the other hand pCURE2N (Figure 2).

In connection with the present invention, the term “gene therapy purpose” is understood to mean that the vector according to the invention, in particular plasmid vector, can be used for any gene therapy purposes, in particular for the introduction of foreign or endogenous nucleic acid sequences, in particular DNA sequences target cells. According to the invention, any cells of the human or animal body, for example a mammalian body, can be understood as target cells. These can be embryonic, stem, body or germline cells of any stage of development. The invention therefore also relates to the use of the vector, in particular plasmid vector, both for somacell gene therapy and for germline gene therapy. However, the invention also covers the use of the vector, in particular plasmid vector, for purposes other than gene therapy, for example for research purposes or for genetic diagnostic purposes.

In connection with the present invention, the term “operatively linked to one another” is understood to mean that the linked elements are linked to one another in such a way that they can interact as intended, for example that operatively linked transcription elements ensure correct transcription. Operatively linked transcription and translation elements enable correct expression, that is to say in particular transcription and translation, to form a functional translation product.

In connection with the present invention, “gene therapy meaningful” nucleotide sequences are understood, for example also genes, gene segments or other structural genome regions, the insertion of which into a target cell makes sense from a therapeutic point of view. The nucleotide sequences to be inserted, in particular genes, can be endogenous or Exogenous to the target cell These can be nucleotide sequences which serve to complement or correct gene defects in the body being treated, but can also be understood to mean one or more gene sections which serve to switch off target genes in target cells Nucleotide sequences, in particular genes or gene segments, which are useful in gene therapy can also be antisense constructs which serve to inhibit transcription in the target cell and thus prevent or reduce the expression of certain proteins.

Genes or gene segments suitable for insertion into the present plasmid vector can of course also be protein-coding or non-protein-coding regions. In connection with the present invention, gene therapy will be under meaningful proteins understood gene products that are expressed in natural form, ie wild-type form, or modified form in target cells after insertion by means of genetic engineering methods.

Proteins of this type which are useful in gene therapy can be any proteins which, for example, occur in defective form in the cell or are not expressed there at all and can be introduced into the cell by means of gene therapy, but can also be proteins which are not naturally present in the target cell occur, but are nevertheless therapeutically desirable there. Such proteins can also be designed as protein fragments or fusion proteins and optionally have modifications, for example post-translational modifications. Of particular interest are genes which code for proteins which are absent in the target cell, are present in reduced amounts or in mutant form. Such proteins can include, in particular, hormones, growth factors, enzymes, lymphokines, cytokines, receptors and the like. In particular, it can be factor VIII, tPA or the molybdenum cofactor.

Molybdenum cofactor deficiency is a severe neurological disease that has not yet been treated and is ultimately fatal. The smallest amounts of intact cofactor and thus also of biosynthesis enzymes are sufficient for a clinically unremarkable phenotype. The main synthesis site is the liver, which is relatively easy to reach. Because of the procedure according to the invention, the enzymes formed here can be used using the set translocation signals are exported and also recorded by the brain.

The present invention therefore also relates to methods for the treatment of molybdenum cofactor deficiency and molybdenum cofactor deficiency diseases, such as the sulfite oxidase deficiency or the xanthine oxidase deficiency.

Further examples of genes to be inserted are genes for hemoglobin, interleukin-1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, GM-CSF, G-CSF, M-CSF, human growth factor , Insulin, factor IX, LDL receptors, tumor necrosis factor, PDGF, EGF, NGF, IL-lra, EPO, TPO, beta-globin and biologically active muteins of these proteins.

Since the plasmid vector according to the invention also leads in particular to the expression of the fusion proteins in the bronchial epithelium, nucleotide sequences are also provided according to the invention in the plasmid vector according to the invention, which code therapeutically useful proteins for the treatment of cystic fibrosis (cystic fibrosis of the lungs). The present invention therefore also relates to a method for the treatment of cystic fibrosis.

In a further preferred embodiment it is provided that the plasmid vector according to the invention contains the constitutive expressing promoter, the human beta-actin promoter, which in particular does not include an intron. In a preferred variant of this embodiment, the human beta-actin promoter comprises the nucleotide sequence SEQ ID No. 10. Ent- speaking of this variant of the promoter region according to the invention, this simplest promoter structure without intron is referred to as plasmid vector pCURE2basic.

In a further embodiment it is provided that the plasmid vector has an expression enhancer, in particular an enhancer, particularly preferably the CMV enhancer. In a further preferred embodiment of the plasmid vector according to the invention, the constitutively expressing promoter comprises in the 5 'to 3' direction a nucleotide sequence coding the CMV enhancer and the human beta-actin promoter. In a preferred variant, the nucleotide sequence encoding the CMV enhancer comprises the nucleotide sequence SEQ ID No. 9.

In a further variant according to the invention, the human beta-actin promoter comprises its native intron. The plasmid vector according to the invention thus provided is referred to as pCURE2natin.

In a further variant, the human beta-actin promoter contained in the plasmid vector according to the invention comprises a shortened intron, the native 5 'and 3' splice sites and a shortened "stuffer ^λλ fragment. In a further variant, the shortened intron of the human beta-actin promoter additionally comprises a nucleotide sequence coding for the SV40 enhancer. According to the invention, the “stuffer” fragment is preferably shortened to such an extent that it only comprises the “branch site”. In particular, the “stuffer” fragment is shortened by 700 to 800 bp; the staged fer fragment shortened by 761 bp. The plasmid vectors according to the invention thus provided are referred to as pCURE2minin (containing the shortened intron) or pCURE2enhin (containing the shortened intron and the SV40 enhancer).

In a preferred embodiment of the plasmid vector according to the invention, the polyadenylation site is the SV40 polyadenylation signal, in a further variant the polyadenylation site is a BGH polyadenylation signal (= BGHpA) of the bovine growth hormone (bovine growth hormone). The polyadenylation site is particularly preferably the BGH polyadenylation signal comprising the nucleotide sequence SEQ ID No. 6.

According to the invention, a ribosome binding site (RBS) with a start codon is located in the 5 'to 3' direction after the constitutively expressing promoter and immediately before the nucleotide sequence encoding a translocation sequence.

According to the invention, a multiple cloning site for the insertion of at least one regulatory element is preferably located in the plasmid vector according to the invention in the 5 'to 3' direction after the constitutively expressing promoter and before the nucleotide sequence coding for a translocation sequence. In a preferred variant of the plasmid vector according to the invention, the at least one regulatory element is a tissue-specific promoter. In a further preferred embodiment, the invention provides that the plasmid vector according to the invention contains at least one resistance gene, in particular a bacterial resistance gene. In a preferred variant, this bacterial resistance gene is the amp ^R gene for ampicilin resistance. In an alternative variant, the bacterial resistance gene is a kan ^R gene for kanaminicin resistance. The kan ^R gene preferably comprises the nucleotide sequence SEQ ID No. 7.

In a further embodiment, the invention provides that the at least one resistance gene has at least one interface for linearizing and / or inactivating the resistance gene.

In a further embodiment, it is preferred that the plasmid vector has at least one origin of replication for plasmid cultivation, particularly in bacteria. In a preferred variant, the origin of replication comprises the nucleotide sequence SEQ ID No. 8.

According to the invention, the plasmid vector is preferably derived from the known vector pcDNA 3.1 (-) and is in particular 3.4 kbp in size (FIG. 1). In a particularly preferred alternative embodiment, the plasmid vector according to the invention is only 2.8 kbp in size and particularly preferably comprises the nucleotide sequence SEQ ID No. 11 (FIG. 2).

In a further embodiment, the invention also relates to host cells or cells of a cell culture containing at least one plasmid vector of the present invention. Such a host cell can be, inter alia, a bacterial cell, a yeast cell, an animal cell, for example a mammalian or insect cell or a human cell.

The invention also relates to methods for the genetic modification of a cell, for example an animal or human cell, comprising contacting the cell with a vector, in particular plasmid vector, according to the present invention under conditions which include an uptake of the vector, in particular plasmid vector allow the cell, especially the cell's genome.

The invention therefore also relates to methods for producing genetically modified cells, the cells to be genetically modified being brought into contact with a vector, in particular a plasmid vector, of the present invention and being transiently or stably transformed.

The invention also relates to the use of a vector, in particular plasmid vector, of the present invention for the production of a preparation, in particular a pharmaceutical preparation for gene therapy, in particular somatic gene therapy.

The invention therefore also relates to preparations, in particular pharmaceutical preparations, containing at least one vector, in particular plasmid vector, of the present invention, optionally together with a pharmaceutically acceptable carrier. Further advantageous embodiments of the invention result from the subclaims.

The invention is explained in more detail with the aid of the following examples, the associated figures and sequence protocols.

Show in the sequence listing

SEQ ID No. 1 the 33 nucleotides encoding the HIV-TAT lead peptide sequence (SEQ ID No. 2),

SEQ ID No. 2 the 11 amino acids of the naturally occurring HIV-TAT lead peptide sequence,

SEQ ID No. 3 the nucleotide sequence containing 49 nucleotides of the region of pCU-RE comprising the RBS, the HIV-TAT region and the NoTl cloning site,

SEQ ID No. 4 the amino acid sequence of the helix of the home domain from Antennipedia,

SEQ ID No. 5 the 11 amino acids of the modified, non-naturally occurring HIV-TAT lead peptide sequence,

SEQ ID No. 6 the nucleotide sequence of the BGH polyadenylation signal containing 235 nucleotides,

SEQ ID No. 7 the nucleotide sequence of the kanamicin resistance gene kan ^R containing 1189 nucleotides, SEQ ID No. 8 the nucleotide sequence of the origin of replication containing 661 nucleotides,

SEQ ID No. 9 the nucleotide sequence of the CMV enhancer containing 348 nucleotides,

SEQ ID No. 10 the nucleotide sequence of the beta-actin promoter containing 377 nucleotides,

SEQ ID No. 11 the nucleotide sequence of the plasmid vector pCURE2basic according to the invention containing 2810 nucleotides (without a cloned nucleotide sequence which makes sense in terms of gene therapy),

SEQ ID No. 12 shows the nucleotide sequence containing 40 nucleotides of the “N-linker” encoding the modified HIV-TAT leading peptide sequence (SEQ ID No. 5), consisting of synthetic oligonucleotides 2NF and 2NR and

SEQ ID No. 13 contains the nucleotide sequence containing the 37 nucleotides of the “C-linker” encoding the modified HIV-TAT lead peptide sequence (SEQ ID No. 5), consisting of synthetic oligonucleotides 2CF and 2CR.

The figures show:

FIG. 1 shows a graphic representation of the plasmid pCURE,

FIG. 2 shows a graphic representation of the plasmid pCURE2basic, FIG. 3 shows an in situ coloring of a brain half transformed according to the invention and a control,

FIG. 4 thin sections of the liver and brain (each negative control and transformed according to the invention),

5 shows thin sections of a brain transformed according to the invention after intrahepatic injection of pCURE2Cbasic-lacZ (FIG. 5A) and thin section of the brain of an untreated control animal (FIG. 5B),

6 shows thin sections of a lung with bronchial epithelium transformed according to the invention after intrahepatic injection of pCURE2Cbasic-lacZ (FIG. 6A) and thin section of the lung of an untreated control animal (FIG. 6B).

Example 1: Preparation of the plasmid pCURE

The expression vector pcDNA 3.1 (-) from Invitrogen (cat. No. V 795-20) was cleaved with EcoRV and Nael. The plasmid was then religated and the nucleotide sequence according to SEQ ID No. 3 was inserted into the NotI site.

FIG. 1 graphically represents the plasmid pCURE in PVU I linearized form (from left to right in 5 'to 3' orientation). The abbreviations used in FIG. 1 mean: P _CMV CMV promoter for cell type-independent expression (source: pcDNA3.1 (-))

MCS multiple cloning site for tissue-specific promoters: NhelU, Pmel, Drall, Apal, Xbal, Xhol

RBS ribosomal binding site with start codon

TAT TAT leader sequence as translocation signal for intercellular transport

Notl insertion site (cloning site) for, for example, therapeutically useful cDNA

SV40pA SV40 polyadenylation signal and transcription termination

pMBl origin of replication ("pUC-derived") for plasmid cultivation in bacteria

ampicillin resistance gene

Pvul interface to linearize and inactivate amplicillin resistance

pCUREl has a size of 3.4 kbp without inserted DNA.

FIG. 1 shows that a multiple cloning site MCS is arranged between P _CM, ie the CMV promoter, and the ribosomal binding site. In the 3 'direction of which follow a TAT leader sequence, a Notl insertion site and the SV40pA polyadenylation site.

Example 2: Preparation of the plasmid pCURE-lacZ

pCURE-lacZ was constructed by cloning the lacZ gene from E. coli into the NotI site of pCURE (Figure 1). The resulting plasmid pCURE-lacZ contains amino acids 8 to 1023 of E. coli beta-galactosidase, which convert Xgal to a blue dye. This catalytic domain lies in the reading frame behind the HIV-TAT translocation leader sequence, which enables intercellular transport.

Example 3: Intraperitoneal application of pCURE-lacZ in mice

pCURE-lacZ was isolated on a mg scale, sterile and pyrogen-free from the E.coli strain JM109 (Qiagen endomaxiprep kit) and after concentration determination adjusted to 150 mM phosphate buffer, pH 7.0 (this buffer also serves as an injection solution for the Control mice).

The injections with pCURE-lacZ were made in healthy mice. Newborn mice were injected with 50 μg pCURE-lacZ in 50 μl phosphate buffer directly into the liver. After 96 hours, the animals were sacrificed and various organs were removed. Brain, liver, heart and lungs were frozen in liquid nitrogen and stored at -70 ° C until further analysis. The above-mentioned organs were incubated in Xgal staining solution (Stratagene Xgal in si tu detection kit) at 37 ° C overnight and used after embedding in paraffin for thin sections of 10-40 μm.

Results :

FIG. 3 shows brain halves stained in total after Xgal incubation. Left: injection of 50 μg pCURE-lacZ. Right: injection of 50 μl phosphate buffer (negative control).

FIG. 4 shows paraffin-fixed thin sections of the corresponding organs, a) and b) liver tissue: a) negative control b) injection of pCURE-lacZ. c) and d) brain: c) negative control d) injection of pCURE-lacZ.

It can be clearly seen that the vectors according to the invention were successfully transferred into the tissue, expressed there and the gene products were also transported into non-transfected organs, in particular the brain.

Example 4: The plasmids pCURE2basic, pCURE2nativ, pCURE2minin and pCURE2enhin

The CMV promoter present on the therapy plasmid pCURE according to Example 1 is replaced by various variants of the human beta-actin promoter. Both promoters are expressed ubiquitously, i.e. in all organs. On the basis of these preliminary experiments, the human beta actin promoter is first used using genomic clones and PCR technologies for use in the therapy plasmids. The human beta-actin gene is ubiquitously expressed for life, thus bypassing promoter inactivation. In order to obtain maximum possible expression rates on the one hand and on the other hand to keep the size of the total plasmid as small as possible - since this in turn directly influences the transfection rates - the human beta-actin promoter is used according to the invention in a total of four different variants of the promoter area:

a) CMV enhancer + beta actin promoter + BGHpA (no intron)

b) CMV enhancer + beta-actin promoter + native beta-actin intron + BGHpA (intron)

c) CMV enhancer + beta actin promoter + truncated beta actin intron + BGHpA (ini intron)

d) CMV enhancer + beta-actin promoter + beta-actin intron, in which the internal intron sequence is replaced by the SV40 enhancer element + BGHpA (enhancer intron)

For a) the "pure" promoter area contains the so-called "CAT" and "TATA" boxes for transcription initiation and represents the smallest of the four possibilities.

For b) this version contains, in addition to the promoter, an intron in front of the start codon or the coding sequence. The intron consists of a 5 'and 3' splice point and an internal "step fer "fragment, which only provides a certain distance between the two splice sites. Since the intron does not contain a coding sequence, it can only have regulatory properties at the level of transcription.

Re c) The mini-intron contains the native 5 'and 3' splice points, but the "stuffer" is greatly shortened in order to keep the size of the total plasmid small. The "stuffer" is shortened so that the so-called " branch site "is still included. In particular, the" stuffer "of the mini-intron is shortened by 761 bp.

Regarding d) The enhancer intron corresponds to the mini intron plus the SV40 enhancer area, which in turn serves as a “stuffer” and thus takes into account a requirement for the distance between the two splice points. On the other hand, it fulfills the passive native “ stuffer "an expression enhancement function. After the transcription, this previously useful area (core import and spacer) is no longer needed and, like the native intron, is spliced out.

Structure of the pCURE2 plasmids:

The promoter area consisting of the CMV enhancer (native sequence from the cytomegalovirus, GenBank M60321), different parts of the human beta actin promoter (native sequence from the human genome, Genbank AC006483), in one of the variants described above from the SV40 enhancer element (na- tive sequence from Simian Virus 40, GenBank NC001669), together with the BGH polyadenylation site (native sequence from the bovine genome, GenBank J00008) forms the expression cassette of the therapy plasmid. This also consists of an origin of replication (ori) for plasmid propagation in bacteria, in particular the native sequence from pUC9 (identical to pUCl3 GenBank L09130) and in particular a kanamycin resistance (native from pACYC177, GenBank X06402) for antibiotic selection in bacteria.

The kanamycin resistance has the further advantage over the ampicillin resistance used in pCURE (example 1) that its use is also approved for clinical trials from phase III. All of the sequence elements of pCURE2 were made individually. A precursor plasmid such as pcDNA3.1 was therefore not used as in pCURE.

In addition, all elements are linked to one another so that a single (unique) Notl interface is always available for cloning in further elements or a coding sequence. This includes the genetic engineering of Notl interfaces in the native beta-actin promoter sequence. The elements linked in this way (expression cassette plus origin of replication plus kanamycin resistance gene) form the therapy vector pCURE2. Corresponding to the four variants of the promoter region described above, pCURE2basic contains the simplest promoter structure a (no intron). pCURE2natin includes Version b (native intron). pCURE2minin includes version c (mini-intron). pCURE2enhin contains version d (enhancer intron).

Use of the translocation sequences:

Experiments carried out at the protein level show that the translocation property of the HIV-TAT protein transduction domain (PTD) can be improved by targeted amino acid exchanges (Ho et al., Cancer Research 2001, 61: 474-477). The best results are obtained from the (non-naturally occurring) sequence YARAAARQARA (SEQ ID No. 5). This sequence is used in the four basic variants of pCURE2 described above, in order to fuse the proteins encoded on the plasmid vector with this domain which causes the translocation property of the fusion proteins.

The four variants of pCURE2 described above are each constructed optionally for the coding of proteins with N- as well as with C-terminal protein transduction domains after the corresponding coding sequences have been cloned into the unique Notl interface. This results in a total of 8 options and therapy plasmids:

pCURE2Cbasic pCURE2Nbasic pCURE2Cnatin pCURE2Nnatin pCURE2Cminin pCURE2Nminin pCURE2Cenhin pCURE2Nenhin

The basal therapy plasmid pCURE2basic with a total of 8 variants is shown in FIG. 2. Preparation of the pCURE2 plasmids:

The sequence for kanamycin resistance (kan ^R ) is amplified from pACYC177 PCR. The origin of replication (ori) is amplified from pUC9 by means of PCR. These two sequences are linked to one another using interfaces which were built into the PCR printer, a unique NotI interface being created according to the invention.

The CMV enhancer sequence, which was amplified from pCURE (example 1) by means of PCR, is incorporated into this, the PCR primers being selected according to the invention such that the emergency (downstream) only in the 3 ^Λ direction from the enhancer (downstream) Interface is restored and thus remains unique.

The BGHpA is amplified from genomic bovine DNA PCR and installed after the enhancer in the unique Notl interface, the PCR primers having been selected according to the invention such that only the Notl interface before the BGHpA (upstream) is restored.

Depending on the promoter version, various areas of the human beta-actin promoter or the SV40 enhancer region, each of which has previously been amplified from genomic DNA, are installed between the enhancer and the polyadenylation site. This installation is carried out according to the principle set forth above, wherein interfaces are so incorporated into the PCR primers that only the 3 ^Λ -NotI interface (downstream) restorers is riert and thus the plasmid can be linearized again with Notl for the next ligation.

According to the same principle, the N- or C-linkers according to the invention are installed, each of which has been completely synthesized. The N-linker according to the invention provides the fusion protein with a N-terminal Proteintranslokationsdomäne (PTD), the 3 ^Λ -NotI interface is preserved for installation of further sequences. According to the invention, the C-linker provides the fusion protein with a C-terminal PTD, the 5 -NotI interface being retained.

Production of pCURE2Nbasic-lacZ and pCURE2Cbasic-lacZ:

The lacZ cassette was isolated for pCURE2Nbasic-lacZ from pCURE-lacZ (Example 2) with restriction enzymes or for PCR for pCURE2Nbasic-lacZ from pCURE-lacZ (Example 2).

Example 5: Intrahepatic application of pCU-RE2Nbasic-lacZ in mice

Two-day-old mice (wild type) were injected intrahepatically with 50 μg of the isolated plasmid vector according to the invention containing the lacZ gene in 50 μl of a 150 mM phosphate buffer with pH 7.0. After 7 days, the animals were sacrificed, the organs removed and, after Xgal staining, paraffin sections of the lungs and brain were made. At the same time, non-injected control animals of the same age were also paraffin sections from Brain and lungs made. The Xgal staining was carried out according to Example 3. All enlargements of the paraffin sections are a hundred times and without counterstaining.

Results:

FIGS. 5A and 5B show completely colored brain halves after Xgal incubation. FIG. 5A: After injection of 50 μg of the plasmid vector. Figure 5B: after injection of 50 ul phosphate buffer (negative control).

The brain sections show on the right the heavily stained choroid plexus and on the left the stained neurons of the cerebral cortex. It can be clearly seen that in the animals treated with the plasmid vector according to the invention, the neurons are colored blue; there the lacZ gene was transformed and expressed according to the invention.

The specific transformation and expression of the marker protein makes it clear that the plasmid vector according to the invention is suitable for the somatic gene therapy of brain neurons. The blood-brain barrier is - particularly surprisingly - overcome by the fusion proteins according to the invention.

Figures 6A and 6B show the results for the lungs: In the lung sections, a bronchus with a strongly stained epithelium can be seen centrally. Due to the lack of staining in the negative control, this structure is difficult to see there. The specific staining of the bronchial pithels shows that the lacZ gene transformed into the bronchial epithelium with the plasmid vector according to the invention is expressed.

The specific staining of the bronchial epithelium makes the use of the plasmid vector according to the invention particularly suitable for treating the cystic fibrosis of the lungs (cystic fibrosis).

Claims

Expectations 1 . Vector for gene therapy purposes, comprising at least one nucleotide sequence encoding a translocation sequence selected from the ^'group consisting of nucleotide sequences encoding the HSV VP22 protein, HIV TAT protein transduction domain, or the Antennipe- dia-peptide. 2nd The vector of claim 1 which is a plasmid vector, a viral vector or a liposome. 3rd A plasmid vector for gene therapy purposes according to claim 2, comprising operatively linked in the 5 'to 3' direction at least one constitutively expressing promoter, the translocation sequence coding nucleotide sequence, a Notl cloning site for the insertion of a gene therapy useful nucleotide sequence and one Polyadenylation site. 4th A plasmid vector for gene therapy purposes according to claim 3, comprising operatively linked in the 5 'to 3' direction the at least one constitutively expressing promoter, a nucleotide sequence coding for a ribosome binding site, the HIV-TAT leader peptide with the amino acids YGRKKRRQRRR (SEQ ID NO. 2) coding nucleotide sequence, a Notl cloning site for the insertion of a gene therapy useful nucleotide sequence and a polyadenylation site. 5. Plasmid vector according to claim 4, wherein the constitutively expressing promoter is the CMV promoter. 6. A plasmid vector according to claim 4 or 5, wherein the polyadenylation site is the SV40pA site. 7. plasmid vector according to any one of claims 4 to 6, wherein between the constitutively expressing Promoter and the nucleotide sequence encoding the ribosome binding site is located a multiple cloning site for the insertion of at least one regulatory element. 8. A plasmid vector according to claim 7, wherein the at least one regulatory element is a tissue-specific promoter. 9. Plasmid vector according to one of claims 3 to 8, wherein the plasmid vector is 3.4 kb in size. 10. A plasmid vector according to one of claims 3 to 9, wherein a nucleotide sequence which is useful in gene therapy is inserted in the Notl cloning site in reading frame for the HIV-TAT lead peptide. 11. plasmid vector according to claim 10, wherein the gene therapy useful nucleotide sequence is selected from the group consisting of Factor VIII gene, tPA gene and molybdenum cofactor Gene. 12. A plasmid vector according to any one of claims 3 to 11, wherein the plasmid vector contains a resistance gene. 13. A plasmid vector according to claim 12, wherein the resistance gene is the amp ^R gene. 14. A plasmid vector according to claim 12 or 13, wherein the resistance gene has at least one interface for linearizing and inactivating the resistance gene. 15. plasmid vector according to any one of claims 3 to 14, wherein the plasmid vector has an enhancer. 16. Plasmid vector according to one of claims 2 to 15, the plasmid vector containing an origin of replication for plasmid cultivation in bacteria. 17. Plasmid vector according to one of claims 2 to 16, wherein the translocation sequence comprises the amino acid sequence of the HIV-TAT lead peptide with the amino acids YGRKKRRQRRR (SEQ ID NO: 2). 18. Plasmid vector according to one of claims 2 to 16, wherein the translocation sequence comprises an amino acid sequence which results from the amino acid sequence SEQ ID NO: 1 by addition, deletion or exchange of at least one amino acid, preferably 6 amino acids. 19. A plasmid vector according to any one of claims 2 to 16, wherein the translocation sequence comprises the modified HIV-TAT leader peptide with the amino acids YARAAARQARA (SEQ ID NO: 5). 20. A plasmid vector according to any one of claims 2 to 16, wherein the translocation sequence comprises a modified amino acid sequence which results from the amino acid sequence SEQ ID NO: 5 by addition, deletion or exchange of at least one amino acid, preferably one to three amino acids. 21. A plasmid vector according to any one of claims 2 to 20, wherein the nucleotide sequence encoding the translocation sequence is contained in a nucleotide sequence functioning as an "N-linker". 22. A plasmid vector according to claim 21, wherein the nucleotide sequence functioning as an "N-linker" comprises the nucleotide sequence SEQ ID NO: 12. 23. A plasmid vector according to any one of claims 2 to 20, wherein the nucleotide sequence coding the translocation sequence is contained in a nucleotide sequence functioning as a "C-linker". 24. A plasmid vector according to claim 23, wherein the nucleotide sequence functioning as a "C-linker" comprises the nucleotide sequence SEQ ID NO: 13. 25. A plasmid vector according to any one of claims 3 to 24, wherein the constitutively expressing promoter is the CMV promoter. 26. A plasmid vector according to any one of claims 3 to 24, wherein the constitutively expressing promoter is the human beta-actin promoter. 27. A plasmid vector according to any one of claims 3 to 24, wherein the constitutively expressing promoter comprises in the 5 'to 3' direction a nucleotide sequence coding for the CMV enhancer and the human beta actin promoter. 28. A plasmid vector according to claim 27, wherein the nucleotide sequence encoding the CMV enhancer is Nucleotide sequence SEQ ID NO: 9. 29. A plasmid vector according to any one of claims 26 to 28, wherein the human beta-actin promoter is characterized in that it does not comprise an intron. 30. The plasmid vector according to claim 29, wherein the human beta-actin promoter comprises the nucleotide sequence SEQ ID NO: 10. 31. A plasmid vector according to any one of claims 26 to 28, wherein the human beta-actin promoter is characterized in that it comprises its native intron. 32. Plasmid vector according to one of claims 26 to 28, wherein the human beta-actin promoter is characterized in that it has a shortened intron, containing the native 5 'and 3' splice sites and a shortened "stuffer" fragment , includes. 33. The plasmid vector according to claim 31, wherein the truncated intron additionally comprises a nucleotide sequence coding for the SV40 enhancer. 34. Plasmid vector according to one of claims 32 or 33, wherein the greatly shortened "stuffer" Fragment is shortened to the extent that it only comprises the "branch site". 35. Plasmid vector according to one of claims 32 to 34, wherein the “stuffer” fragment is shortened by 700 to 800 bp. 36. Plasmid vector according to one of claims 32 to 34, wherein the "stuffer" fragment is shortened by 761 bp. 37. Plasmid vector according to one of claims 3 to 35, wherein the polyadenylation site the SV40- Is polyadenylation signal. 38. Plasmid vector according to one of claims 3 to 37, wherein the polyadenylation site is a BGH polyadenylation signal. 39. A plasmid vector according to claim 38, wherein the BGH polyadenylation signal comprises the nucleotide sequence SEQ ID NO: 6. 40. A plasmid vector according to any one of claims 3 to 39, wherein a ribosome binding site with a start codon is located in the 5 'to 3' direction after the constitutively expressing promoter and immediately before the nucleotide sequence coding for a translocation sequence. 41. A plasmid vector according to one of claims 3 to 40, wherein a multiple cloning site for the insertion of at least one regulatory element is located in the 5 'to 3' direction after the constitutively expressing promoter and before the nucleotide sequence coding for a translocation sequence. 42. A plasmid vector according to claim 41, wherein the at least one regulatory element is a tissue-specific promoter. 43. A plasmid vector according to any one of claims 3 to 42, wherein the plasmid vector contains at least one resistance gene. 44. A plasmid vector according to claim 43, wherein the at least one resistance gene is the amp ^R gene for ampicillin resistance. 45. A plasmid vector according to claim 43, wherein the at least one resistance gene is a kan ^R gene for camicin resistance. 46. A plasmid vector according to claim 45, wherein the kan ^R gene comprises the nucleotide sequence SEQ ID NO: 7. 47, plasmid vector according to any one of claims 43 to 46, wherein the at least one resistance gene has at least one interface for linearizing and inactivating the resistance gene. 48. Plasmid vector according to one of claims 3 to 48, wherein the plasmid vector contains at least one origin of replication for plasmid cultivation, particularly in bacteria. 49. A plasmid vector according to claim 48, wherein the at least one origin of replication comprises the nucleotide sequence SEQ ID NO: 8. 50. Plasmid vector according to one of claims 4 to 49, the plasmid vector being derived from the vector pcDNA3.1 (-). 51. The plasmid vector according to claim 50, wherein the plasmid vector is 3.4 kbp in size 52. Plasmid vector according to one of claims 3 to 49, wherein the plasmid vector is 2.8 kbp in size. 53. A plasmid vector according to any one of claims 3 to 51, wherein the plasmid vector contains the nucleotide sequence SEQ ID NO: 11. 54. A plasmid vector according to any one of claims 3 to 53, wherein a nucleotide sequence which is useful in gene therapy is inserted in the NotI cloning site in the reading frame for a translocation sequence. 55. A plasmid vector according to claim 54, wherein the nucleotide sequence which is useful in gene therapy is selected from the group consisting of factor VIII gene, tPA gene and molybdenum cofactor gene. 56. Host cell containing a plasmid vector according to one of claims 2 to 55. 57. The host cell according to claim 56, wherein the host cell is a bacterial cell, an insect cell, a mammalian cell or a human cell. 58. A method of genetically modifying a cell, comprising contacting the cell with a vector according to any one of claims 1 to 55 under conditions suitable to effect the uptake of the vector into the cell. 59. A method of introducing genetic information into a cell, comprising inserting the genetic information into a vector according to any one of claims 1 to 55 and contacting the cell with the plasmid vector under conditions suitable for incorporating the genetic information into the cell to effect. 60. The method according to any one of claims 58 or 59, wherein the cell is a bacterial, human or animal cell. 61. A pharmaceutical composition containing a vector according to any one of claims 1 to 55, optionally together with a pharmaceutically acceptable carrier. 62. Use of a plasmid vector according to one of claims 1 to 55 for the production of a preparation for somatic gene therapy. 63. Use according to claim 62 for the manufacture of a preparation for the treatment of a molybdenum cofactor deficiency. 64. Use according to claim 62 for the manufacture of a preparation for the treatment of cystic fibrosis.