DE102005003207A1 - Lentiviral gene delivery system for ribozyme-mediated RNA repair - Google Patents

Abstract

The invention relates to a recombinant lentiviral gene transfer system for ribozyme-mediated repair of a target RNA comprising: DOLLAR A a. a first plasmid containing a nucleic acid sequence coding for the polyprotein gag of the visna lentivirus (VLV), said plasmid not coding for other VLV proteins and containing no competent packaging signal, DOLLAR A b. a second plasmid as a transfer plasmid, containing: DOLLAR A (i) VLV nucleic acid sequences which contain the cis-acting sequence elements required for the reverse transcription of the plasmid genome, DOLLAR A (ii) a competent packaging signal (packaging signal ) and DOLLAR A (iii) a ribozyme cassette containing the sequence of the catalytic domain of a self-splicing intron ribozyme (Group I intron) and a heterologous gene or portion of a gene encoding the accurate counterpart of the target RNA to be repaired , or a cloning site into which a heterologous gene or part of a gene coding for the accurate counterpart of the target RNA to be repaired can be inserted, DOLLAR A c. a third plasmid containing a nucleic acid sequence encoding the VLV polyprotein Pol, which plasmid does not encode other VLV proteins and does not contain a competent packaging signal, DOLLAR A d. a fourth plasmid containing nucleic acid sequences coding for the VLV proteins Vif, Rev and Tat, wherein the plasmid does not encode other VLV proteins and ...

Description

The The invention relates to viral vectors useful for gene transfer and gene therapy are suitable, in particular lentiviral vectors, which are for the ribozyme-mediated RNA repair in non-dividing and dividing cells suitable.

The Ability, a particular foreign or native gene sequence in a mammalian cell to introduce and control the expression of the gene is of great importance for the medical and biological research. This ability provides a means to available which allows to study gene regulation and a therapeutic one base for to develop the treatment of diseases.

Gene Therapy It is typically designed to provide a mutant allele of a gene a functional supplement. Although the technology is still in its infancy, was they already used with some success. However, most gene therapy approaches add just add a functional allele and replace the flawed mutant Allele of the gene is not. As a result, the mutant allele remains intact - which is not Problem if the mutant allele is not active. If however, if the mutant allele has altered activity, they will approaches fail because they only one desired activity next to a changed one activity put. Another disadvantage of conventional gene therapy approaches is that the level of expression of the inserted gene is very much on the Location of the genome depends in which the gene is inserted. This is different Expression level usually from the natural level of expression of Gene.

Among the viral vectors used for gene transfer, retrovirus-based vectors are particularly preferred because they provide stable integration of the transferred gene into the genome of mammalian cells. Lentiviruses are a subset of retroviruses that can infect cells that do not divide. Several lentiviral vector systems based on the human immunodeficiency virus (HIV) or the (closely) related simian immunodeficiency virus (SIV) are described ( US 5,665,577 ; US 5,747,324 ; US 5,861,282 ; US 5,919,458 ; US 5,981,276 ; US 6,013,516 ; US 6,555,342 ; US 6,627,442 ; US 6,669,936 ; US 6,712,612 ; US 6,790,641 ; US 6,790,657 ).

However, the use of HIV or SIV-based systems for gene transfer into humans poses serious safety issues as the possibility of recombination of the vector into a virulent and disease-causing form exists. This is less true for vectors based on related viruses, such as the Bovine Immunodeficiency Virus (BIV - WO 03/066810), the Equine Infectious Anemia Virus (EIAV). US 6,277,633 . US 6,312,683 ) or the Feline Immunodeficiency Virus (FIV - US 6,555,107 ). The pronounced homology between the helper and structural protein genes and the transduction vector (s) used in these approaches increases (but increases) the probability of recombination.

US 6,479,281 relates to lentiviral virus vectors (derived from HIV, SIV, FIV, Visnavirus or EIAV) which have a truncated matrix protein (claim 17b) which has been replaced with a heterologous myristylation anchor and a truncated coat protein (envelope protein - claim 17c). Since the vector is a replication-competent retrovirus, the risk of spread and recombination is considerable. No examples of transduction of a heterologous gene are shown.

The Visnavirus (Virus Code 61.0.6.4.002) is a lentivirus, the sheep infected and for People is not pathogenic. The wild type visnavirus has a dimeric RNA genome (single-stranded, positive polarity), which in a spherical, sheathed Virion, which contains a nucleoprotein core. The Replication of the wild-type visnavirus genome is by reverse Transcription and integration into the genome of the host cell. The virus genome consists of an RNA (9202 bp, Genbank Accession Number: NC 001452) and contains three essential genes for which encode the gag, pol and env polyproteins, and long terminal repeats (LTR) at each end of the integrated viral genome. The LTR is in about 600 nt long, of which the U3 region is 450, the R sequence 100 and the U5 region about 70 nt long. The gag gene of the wild-type Visnavirus coded for the polyprotein gag, which is transformed by the viral protease into the structural protein the matrix p17, the major capsid protein p24 and cleaving the nucleocapsid proteins p7 to p11. The pol Gene codes for the polyprotein Pol, which passes through the protease into the following Enzyme is cleaved: the protease itself, the reverse transcriptase (RT), RNase H (RH), dUTPase and integrase.

The natural visnavirus contains additional genes that code for the helper proteins Vif, Tat, and Rev, which are involved in the regulation of synthesis and processing of viral RNA and other replicative functions. The Vif (Viral Infectivity Factor) protein is packaged with the integrase portion of the pre-integration complex, which encases the DNA product that is formed by the viral RNA through the Pol-derived RT in a form that it allows him to pass through the nuclear membrane. Thereby, the stable integration of the viral genome into the host cell genome in the presence of an intact nuclear membrane is possible, such. In a non-dividing (resting) or finally differentiated cell. The Tat (transactivator of transcription) protein is a pleiotropic factor that induces a range of biological effects in a variety of cell types. Tat is a powerful gene expression transactivator that exerts its effect by inducing chromatin rearrangement as well as by recruitment of elongation-competent transcriptional complexes to the viral LTR promoter. The visnavirus Tat protein is required for the efficient viral transcription of the visnavirus LTR (long terminal repeat). AP-1 regions within the visnavirus LTR, to which the cellular transcription factors Fos and Jun bind, are also necessary for Tat-mediated activation of transcription.

The Contains wild-type genome of the visnavirus also several cis-acting sequences, promoter elements such as the Tat Response Element (TRE), which begins the transcription process control the integrated provirus; a packaging sequence (Y); and a Rev-Response Element (RRE), which is responsible for the export of virion mRNA from the host cell core is responsible.

The Disadvantages of most retroviruses, including the wild type visnavirus, are a limited cell tropism, a low virus titer and the risk of generating replication competent viruses during the Packaging.

One Another problem with traditional gene therapy approaches is that when a healthy gene is inserted, it is a defective gene to replace it randomly inserted into the host genome becomes. This can cause problems because the gene has a lot of its (usually) it loses surrounding DNA, which is regulatory Contains information; The substitute gene used can also disturb his new neighbors.

One different approach to gene therapy is antisense therapy, which aims to shut down a mutant allele of a gene, by being complementary nucleic acid sequences to be inserted.

ribozymes are RNAs that have enzymatic activity. different Ribozymes are known, such as the hammerhead ribozyme, the hairpin ribozyme, the Tetrahymena group I intron, RNase P and the hepatitis delta virus ribozyme. They catalyze an internal cut (self-cleavage - intramolecular or "in-cis" catalysis) or they cut external substrates (intermolecular or "in-trans" catalysis). Ribozymes, like the hammerhead ribozyme and the hairpin ribozyme, are already in lentiviral vectors for genes silencing approaches (e.g., WO97 / 10334, WO0032765). In the gene silencing approaches will be Ribozymes using many of the known from the antisense nucleic acids Principles used. Ribozymes are also based on complementary binding of nucleic acids to recognize their target molecule (Target). Unlike antisense nucleic acids, however, they do not bind only to a target messenger RNA, but also silences the gene, by cutting the RNA.

Of the Group I intron-ribozyme-mediated mechanism of RNA repair has long been considered a promising tool for gene therapy (summarized by Long MB et al., 2003 J. Clin. Invest. 112: 312-318). The use of group I intron ribozyme mediated RNA repair for However, gene therapy has so far been limited by the efficiency of Transfers of for the ribozyme-encoding gene in human cells, the limited intracellular activity of the ribozyme in the cytoplasm and the short useful life of the ribozyme transient transfection difficult.

To today is not a safe and efficient gene transfer system for the ribozyme-mediated RNA repair is known for a big Bandwidth of dividing and non-dividing cells used can be. Because the safety of gene transfer is mediated by the packaging constructs of the highest Interest, there is still a need for safer and efficient lentiviral vector systems that transfer gene into one size Bandwidth of dividing and mediating non-dividing cells can.

A Object of the present invention is therefore a safer and Efficient Gene Transfer System for Ribozyme-Mediated RNA Repair indicate that in a large Bandwidth of dividing and non-dividing cells can be.

• a. ein erstes Plasmid enthaltend eine Nukleinsäuresequenz codierend für das Visna-Lentivirus (VLV) Polyprotein Gag, wobei das Plasmid nicht für andere VLV-Proteine codiert und kein kompetentes Verpackungssignal (packaging signal) enthält,
• b. ein zweites Plasmid als Transfer-Plasmid enthaltend: (i) VLV-Nukleinsäuresequenzen, welche die für die reverse Transkription des Plasmidgenoms benötigten Cis-acting Sequenzelemente (cis-acting sequence elements) enthalten, (ii) ein kompetentes Verpackungssignal (packaging signal) und (iii) eine Ribozymkassette, welche die Sequenz der katalytischen Domäne eines selbst-spleißenden Intron-Ribozyms (Gruppe I Intron) enthält, und (iv) ein heterologes Gen oder ein Teil eines Gens, welches für das akkurate (nicht-mutierte) Gegenstück der zu reparierenden Target-RNA codiert, oder eine Klonierungsstelle (cloning site), in die ein heterologes Gen oder ein Teil eines Gens, welches für das akkurate (nicht-mutierte) Gegenstück der zu reparierenden Target-RNA codiert, eingesetzt werden kann, wobei das zweite Plasmid nicht für irgendein funktionelles Protein des VLV codiert;
• c. ein drittes Plasmid enthaltend eine Nukleinsäuresequenz, die für das VLV-Polyprotein Pol codiert, wobei das Plasmid nicht für andere VLV-Proteine codiert und kein kompetentes Verpackungssignal (packaging signal) enthält,
• d. ein viertes Plasmid enthaltend Nukleinsäuresequenzen codierend für die VLV-Proteine Vif, Rev und Tat, wobei das Plasmid nicht für andere VLV-Proteine codiert und kein kompetentes Verpackungssignal enthält,
• e. ein fünftes Plasmid enthaltend eine Nukleinsäuresequenz codierend für ein virales Hüllprotein (envelope protein – Env), welches nicht vom Visnavirus abgeleitet ist und wobei das Plasmid nicht für VLV-Proteine codiert und kein kompetentes Verpackungssignal enthält.

This object is achieved according to the invention by a recombinant lentiviral gene transfer system for the ribozyme-mediated repair of a mutated target RNA comprising:

• a. a first plasmid containing a nucleic acid sequence encoding the Visna lentivirus (VLV) polyprotein gag, which plasmid does not encode other VLV proteins and does not contain a competent packaging signal,
• b. a second plasmid as a transfer plasmid containing: (i) VLV nucleic acid sequences which contain the cis-acting sequence elements required for the reverse transcription of the plasmid genome, (ii) a packaging signal and iii) a ribozyme cassette containing the sequence of the catalytic domain of a self-splicing intron ribozyme (group I intron), and (iv) a heterologous gene or part of a gene coding for the accurate (non-mutated) counterpart of cloning site) into which a heterologous gene or part of a gene coding for the accurate (non-mutated) counterpart of the target RNA to be repaired can be inserted, the second Plasmid not encoded for any VLV functional protein;
• c. a third plasmid containing a nucleic acid sequence coding for the VLV polyprotein Pol, which plasmid does not encode other VLV proteins and does not contain a packaging signal,
• d. a fourth plasmid containing nucleic acid sequences encoding the VLV proteins Vif, Rev and Tat, wherein the plasmid does not encode other VLV proteins and does not contain a competent packaging signal,
• e. a fifth plasmid containing a nucleic acid sequence coding for a viral envelope protein (Env), which is not derived from Visnavirus and wherein the plasmid does not encode VLV proteins and does not contain a competent packaging signal.

• 1. Aufteilung (Separierung) der Gene, welche für die funktionellen VLV-Proteine Gag, Pol, Vif, Tat und Rev codieren, auf drei unterschiedliche Plasmide,
• 2. Abtrennen der Gene, welche für die funktionellen VLV-Proteine codieren, von dem Verpackungssignal und den für die Reverse Transkription benötigten Cis-acting Sequenzen (cis-acting sequences),
• 3. Abtrennen der Gene, welche für die funktionellen VLV-Proteine codieren, von dem für das Hüllprotein (envelope Protein) codierenden Gen, welches nicht vom Visnavirus abgeleitet ist.

The recombinant lentiviral gene transfer system according to the invention can be used to produce replication-deficient virion vector particles. These are a useful tool for ribozyme-mediated gene therapy. The safety of the recombinant lentiviral gene delivery system is improved as a combination of several strategies minimizes the risk of recombination and functional virus formation. The combined strategies are:

• 1. Separation of the genes coding for the functional VLV proteins Gag, Pol, Vif, Tat and Rev on three different plasmids,
• 2. separating the genes coding for the functional VLV proteins from the packaging signal and the cis-acting sequences required for the reverse transcription,
• 3. Separating the genes coding for the functional VLV proteins from the envelope protein coding for the envelope protein, which is not derived from the Visnavirus.

The Risk of recombination is further reduced and safety further increased, because the genes, which for encoding the functional proteins of the visnavirus, preferably synthetic, non-naturally occurring nucleotide sequences are constructed.

The Proteins Pol, Vif, Rev and Tat are for the proper function of the recombinant lentiviral gene transfer system.

Around to reduce the risk of occurrence of recombination events, are all plasmids of the invention designed to have minimal homology at the nucleic acid level and minimal overlaps to each other.

The for the Pol, the accessory (Vif, Rev and Tat) and the structural (Gag) protein coding sequences are from the transfer plasmid separated and distributed to 3 separate plasmids.

By the division of the construct genome on a number of plasmids will increase the risk of producing replication-competent retroviruses starting from the gene transfer construct according to the invention decreased as the number of necessary recombination events elevated becomes.

in the Unlike the natural Visnavirus, in which the gag and pol genes overlap, are the genes according to the invention, the for the nucleocapsid protein Gag and the Pol protein encode two distributed different plasmids. These two plasmids will be hereinafter referred to as "Pol plasmid" (c) and "Gag plasmid" (a).

In wild-type virus, the gag and pol polyproteins are separated by overlapping open reading frames co in a way that ensures that Pol is formed at a lower rate than Gag.

in the inventive gene transfer construct The gag and pol proteins are derived from synthetic nucleotide sequences coded. The synthetic nucleotide sequences code for the same amino acid sequences as in wild type, but use codons that are so different as possible of the wild type sequences. This change in codon usage eliminates the possibility an overlap the ORFs (open reading frames) of Gag and Pol coding Sequences.

Around should ensure a high titer of the gene transfer construct both the gag as well also the pol expression under the control of strong promoters be. The for However, gag and pol coding sequences must be under the control be from different promoters. In the wild type visnavirus becomes the pol polyprotein at a rate (approximately 20 times) lower than the gag polyprotein. As in the invention, the for Pol coding sequence and the for Gag coding sequence are distributed to different plasmids, Their products should also be made in different rates in a relationship, which the natural relationship equivalent. In order to achieve this, the pol expression is always according to the invention under the control of a weaker, preferably a ten to twenty times weaker promoter than Gag expression. Preferably, the for Gag coding sequence under the control of cytomegalovirus (CMV) promoter and the for Pol coding sequence under the control of simian virus 40 (SV 40) promoter. Other promoter pairs that make sure the Gag expression significantly exceeds pol expression, are also suitable.

The for the accessory proteins of the visnavirus coding genes, Vif, Rev and deed that for the production of virions are necessary are preferred on one located third plasmid, which is hereinafter called "VTR plasmid" (d).

in the Wildtype virus overlaps the vif ORF (open reading frame) with the pole ORF in 43 base pairs (Bp). In the present invention, the change in the codon usage of vif and pol t that overlap between these ORFs is suspended and that the ORFs are under control two different promoters are. This is achieved by the sequences for encode the accessory proteins Vif, Tat and Rev to a separate Plasmid be spun off. Again, the separation of the sequences leads to vif, did and rev from the sequence pol to improve security.

The Gene transfer system contains a transfer plasmid (b) with a cassette for the group I intron ribozyme mediated RNA repair. This cassette contains a Group I intron ribozyme sequence and a cloning site into which a heterologous gene or part of a heterologous gene is inserted can be.

If the transfer plasmid is transferred to a eukaryotic cell, The group I intron ribozyme sequence is stable as DNA in the genome the target cell integrated, where the ribozyme RNA under the control of a viral promoter is expressed. The ribozyme sequence and the heterologous Gene sequence are then rewritten into an RNA that binds to the mRNA anneals, which is expressed by a target gene (Target mRNA). This ribozyme then splices a predefined one Sequence 3 'of one Section which makes the ribozyme into the target mRNA. By the trans-splicing activity of the group I intron ribozyme a portion of the target mRNA exchanged by the RNA, the provided by the heterologous gene sequence.

Compared with conventional gene therapy approaches, this group has I intron Ribozyme-mediated RNA repair has the advantage that the target is under the control of his natural Promoter is transcribed and subsequently at the messenger RNA level is repaired. By the trans-splicing activity of the group I intron ribozyme is the mutant portion of the target mRNA the faultless counterpart replaced. In this way, the target mRNA becomes an RNA again together, the for encodes a functional protein having the desired activity.

• 1. der Promotor, welcher die Expression des Ribozyms steuert;
• 2. die Leitsequenz (guide sequence – GS), welche verantwortlich ist für die Erkennung der Target-mRNA und der Schnitt- und Spleiß-Stelle innerhalb der Target-mRNA;
• 3. die katalytische RNA;
• 4. die Sequenz, die 3' auf den geschnittenen 5'-Anteil der Target-mRNA gespleißt wird, oder eine Klonierungsstelle, in welche die Sequenz eingesetzt werden kann, die 3' auf den geschnittenen 5'-Anteil der Target-mRNA zu spleißen ist.

The ribozyme cassette, which contains the group I intron catalytic ribozyme sequence, has four important functional domains:

• 1. the promoter which controls the expression of the ribozyme;
• 2. the guide sequence (GS), which is responsible for the recognition of the target mRNA and the cut and splice site within the target mRNA;
• 3. the catalytic RNA;
• 4. the sequence spliced 3 'to the cleaved 5' portion of the target mRNA, or a cloning site into which the sequence can be inserted, 3 'to the cleaved 5' portion of the tar to splice get mRNA.

The Ribozyme cassette thus consists of the promoter, which is the expression of the ribozyme, followed immediately by the leader, which the target mRNA recognizes and binds, followed by the catalytic Unit of ribozyme followed by the sequence 3 'to the 5' portion of the cut Target mRNA molecule spliced becomes.

The Expression of the ribozyme is preferably under the control of SV40 Promotors posed.

The The catalytic unit of the ribozyme is preferably from a Tetrahymena thermophylia 26S rDNA intron, preferably T. hyperangularis (GenBank X03106), T. sonneeborni (GenBank X03108), T. cosmopolitanis, (GenBank X03107), T. pigmentosa (GenBank V01412) particularly preferably T. thermophylia (GenBank V01416).

The Guide sequence and the to be spliced Sequence become dependent selected from the target mRNA molecule. The Guide sequence goes directly to the catalytic unit of the ribozyme 3 'ahead and recognizes the defined splice site in the target RNA by Watson-Crick base pairing.

The Sense oligonucleotide of the DNA fragment contains the sense portion of the leader followed by the first 5 nucleotides of the catalytic ribozyme sequence. The opposite (reverse) complementary oligonucleotide of the DNA fragment contains the 5 nucleotide complement to the overhang at the end of the ribozyme promoter sequence followed by the antisense complement to the desired leader sequence.

The Sequence, the 3 'up the cut 5 'portion the target mRNA spliced will depend on the place of the mutation that needs to be repaired. As the trans-splicing activity of the ribozyme the 3'-range of Exchanges target mRNA containing the ribozyme cassette contains the nucleotides corresponding to the nucleotide residues, where the mutation occurred and the area of the target mRNA coding gene, in the 3 'direction from the nucleotide residues in which the mutation occurred is up to the end of said gene.

The Ribozyme cassette is preferably constructed as a unit. From One unit of ribozyme cassette is suitable for another Development as a therapeutic agent for the correction of mutated RNA.

A preferred ribozyme cassette for repair of the mutated amyloid precursor protein (βAPP) mRNA is part of the transfer plasmid with the nucleotide sequence according to SEQ ID no. 6 and the in 4 shown plasmid map. This ribozyme cassette is designed to repair a series of mutations in the mRNA encoding the amyloid precursor protein (βAPP).

Prefers the L1 loop of the catalytic unit of the ribozyme is so altered that he has an opposite (reverse) complement (here the 6-nucleotide sequence GGACAT) to the 5 'portion the second to seventh nucleotide, the sequence to be spliced (here: ATGTCC) has. This approach can be applied to any mutated sequence belonging to the genome of the Nucleus is encoded to exchange. The reverse complement is Part of the guide sequence "GS".

A preferred, for the ribozyme used leader sequence (GS, which contains the amyloid precursor protein (βAPP) mRNA detects which of both NGF-stimulated PC12 cells and HEK293 is GGTGGCGCTCCTCTGGGG. This GS serves as a Detection area and splice site for the Ribozyme. The GS-DNA fragment is for example composed of two oligonucleotides GCTCGGTGGCGCTCCTCTGGGG and TAATCCCCAGAG-GAGCGCCACC. An annealing of these two oligonucleotides produces a DNA fragment which for the anti-APP-ribozymale GS and is compatible to the 5'-overhang of the ribozyme and the 3'-overhang of the CMV promoter. Changing the sequence of the GS allows the ribozyme practically adapt to any target mRNA.

In another embodiment of the invention, the ribozyme cassette contains a stuffer sequence instead of the guide sequence The leader sequence or sequence has a preferred length of 5 to 20 nucleotides, more preferably 10 to 16 nucleotides in length The filling sequence allows the end user to insert a guide sequence (GS) of his choice into the ribozyme cassette The target RNA and the splice site within the target RNA are determined by the GS for the ribozyme The transfer plasmid with a filling sequence instead of the leader sequence is thus a versatile construct that can be tailored to correct for the target RNA of choice is designed to facilitate their exchange for a leader sequence. The fill sequence contains unique (unique) recognition sites for restriction sites. The filling sequence which replaces the leader sequence, e.g. Gcgcgccgaacctcgagtacgccggcg, preferably contains a recognition site for the restriction enzyme Psrl. The PsrI restriction site ([7/12] GAACNNNNNNTAC [12/7]) is cut both once up and down once from the restriction enzyme recognition site, resulting in 5-nucleotide overhangs.

Of the End user replaces the fill sequence, a transfer plasmid with the specificity for a target RNA of choice, which is to be repaired or edited. The filling sequence becomes replaced by a DNA fragment containing the leader sequence. These is composed of two oligonucleotides, which are preferred 10 to 20 nucleotides, more preferably 12 to 16 nucleotides, are long.

In another embodiment of the invention, the ribozyme cassette is constructed with a sequence encoding a marker protein and spliced 3 'onto the cut 5' portion of the target RNA. Preferred marker proteins are the Enhanced Green Fluorescent Protein (EGFP), Placental Alkaline Phosphatase (PLAP) or human codon-optimized versions of the nucleic acids encoding E. coli β-galactosidase or E. coli alkaline phosphatase (ECAP). These ribozyme cassette sequences are part of the transfer plasmids pBR322dtVLV-ECAP, pBR322dtVLV-PLAP, pBR322dtVLVt-βGal and pBR322dtVLV-GFP (SEQ ID Nos. 7 to 10, 2 and 9 to 11 ).

The Ribozyme cassette containing a filling sequence, instead of the leader sequence and contains a marker protein which spliced to the target RNA can be used to a series of transfer plasmids that hybridize a marker protein to an endogenous protein, which is normally expressed by the cell. These hybrid proteins are suitable for Transport and localization studies.

An example of a ribozyme cassette with a filling sequence instead of the leader sequence and a sequence coding for the marker protein GFP is part of the transfer plasmid pBr322dtVLV-GFP having the nucleic acid sequence according to SEQ ID. 10 and the in 11 shown plasmid map.

In a preferred embodiment In the invention, the ribozyme cassette is constructed with a filling sequence instead of the leader sequence and a cloning site instead of the Sequence, the 3 'up the cut 5 'section the target RNA spliced becomes.

If the sequence, the 3 'on the cut 5 'section the target RNA spliced is replaced by a cloning site, this contains Cloning site (eg aggtcctagtactcgagtacctgcaggg) preferred a recognition agency for the restriction enzyme Bael ((10/15) ACNNNNGTAYC [12/7]). Restriction Digest with Bael leaves a 5 nucleotide overhang at the 3'-end of the catalytic sequence of the ribozyme and a 5'-overhang at the 5 'end of the U3 sequence. One for a marker protein coding sequence can be between these two Jobs are advertised.

A Ribozyme cassette with a filling sequence (stuffer) instead of the guide sequence and a cloning site instead of the sequence, the 3 'on the cut 5 'portion of the target RNA spliced is a versatile construct that in principle is so adapted It can be specific to any target RNA of interest cuts and splices.

An example of a ribozyme cassette having a fill sequence in place of the leader sequence and a cloning site in place of the sequence spliced to the target RNA is in the transfer plasmid pBR322dtVLV-PsrBae having the nucleic acid sequence of SEQ ID NO. 5 ( 12 ) contain.

The Contains transfer plasmid further cis-acting elements (cis acting elements) responsible for reverse transcription of the plasmid genome needed and a competent packaging signal. Cis-acting elements are RNA regions, which as RNA play a functional role in processing of the plasmid or cassette in which they are contained.

The Contains transfer plasmid prefers the main cis-acting elements used for packaging of the viral genome responsible for the virion. These are in the wild-type visnavirus and in the gag transfer plasmid 3 'and gag in the p16 region localized. Gag was inactivated in the transfer plasmid of the invention, by removing the start codon (SEQ_ID No. 16) and adding it was truncated behind the p16 homologous region to D-p16 obtained (SEQ ID No. 17). This procedure inactivates gag and lets the cis-acting elements intact.

• 1. dem Rev response element (RRE, SEQ_ID No. 21) des Wildtyp-Visnavirus,
• 2. der primer-binding site des Wildtyp-Visnavirus,
• 3. den LTRs des Wildtyp-Visnavirus,
• 4. dem Polypurinabschnitt (poly purine tract) des Wildtyp-Visnavirus und seinem Komplement,
• 5. der zentralen Terminationssequenz (termination sequence) des Wildtyp-Visnavirus,
• 6. inaktivierten Sequenzen von VLV tat (D-tat, SEQ_ID No. 18), VLV rev (D-rev, SEQ_ID No. 19) und VLV env (D-env, SEQ_ID No. 20), die nicht für funktionale Proteine codieren, da die Startcodons entfernt sind.

While the key portions of the RNA elements responsible for interacting with the virion packaging proteins are localized in the gag leader sequence Gag-L and in D-p16, they are not exclusive to these positions limited. Therefore, the transfer plasmid preferably contains further cis-acting elements selected from:

• 1. the rev response element (RRE, SEQ ID 21) of the wild-type visnavirus,
• 2. the primer-binding site of the wild type visnavirus,
• 3. the LTRs of the wild-type visnavirus,
• 4. the polypurine section (poly purine tract) of the wild-type visnavirus and its complement,
• 5. the central termination sequence of the wild-type visnavirus,
• 6. inactivated sequences of VLV tat (D-tat, SEQ ID 18), VLV rev (D-rev, SEQ ID 19) and VLV env (D-env, SEQ ID 20) which do not encode functional proteins because the start codons are removed.

The Cis-acting elements are preferably selected from the group of nucleotide sequences according to SEQ_ID No. 16 to 21.

The competent packaging signal (packaging signal) in the transfer plasmid includes RNA elements that are sufficient to wrap the packaging of the RNA molecule into a To induce virion. The competent packaging signal is included even a part of the RNA molecule to be packaged and in the gag leader (Gag-L) and in the inactivated p16 (D-p16) Sequence of the transfer plasmid included.

All other plasmids (all except the transfer plasmid) do not have a competent packaging signal. The is preferably achieved by the packaging signal in the design the artificial one Sequences is omitted.

The Transfer plasmid containing cis-acting elements coded not for functional proteins of the VLV. The lack of functional proteins in the transfer plasmid and the lack of homology with the plasmids, which contain the functional proteins eliminate the risk of that from the gene transfer construct a recombinant, for replication capable Virus could emerge.

preferred Transfer plasmids have the sequences according to SEQ ID no. 5 to 10.

Around to remove the cis-acting elements from the Gag plasmid of the invention, the area became 3 'of gag away. In the Gag plasmid according to the invention The cis-acting elements were gag by maximum change destroyed the codon usage. The expression of Gag in the Gag plasmid according to the invention is preferred under the CMV promoter. The gag plasmid contains thus no functional packaging signal and is unable to to recombine with the inactivated gag sequence on the transfer plasmid.

In the recombinant according to the invention lentiviral gene transfer system was the gene responsible for the visnavirus coat protein (envelope protein), inactivated by omitting the start codon was to get D-env. The wild-type env ORF contains the rev1 and rev2 ORFs and RRE. A changed form rev is contained in the VTR plasmid. To the cell tropism of the To expand gene transfer constructs in the invention is the gene for the Visna virus coat protein replaced by a gene which is responsible for a coat protein (envelope protein) encoded by another virus. The procedure to envelop the gene transfer constructs with an envelope protein which from the wild-type virus is called pseudo-typing. preferred envelope proteins are: Ebola Envelope (GenBank U31033), gp64 Envelope Glycoprotein of Bakulovirus (GenBank AF190124), influenza A-haemagglutinin (Gen Bank, Z46395), Jaagsiekte sheep retrovirus (JSRV) Envelope (GenBank Y18303), Lymphocytic Choriomeningitis virus (LCMV) glycoprotein (LCMV-GP, GenBank AF186080), Mokolavirus Envelope Glycoprotein (MK-G, GenBank S59448), Amphotropic Murine Leukemia Virus (MuLV) Amphotropic envelope (4070A-Env, Genbank M33469), Rabies virus envelope glycoprotein (GenBank AB110669), RD114 retrovirus envelope (RD114 Env; GenBank X87829). A particularly preferred envelope protein is the vesicle Stomatitis virus envelope glycoprotein (VSVG; GenBank V01214).

The envelope protein (Env) coding sequence is preferably located on another plasmid (s) which does not encode VLV proteins and does not contain a competent packaging signal. This plasmid is hereinafter referred to as "Env plasmid." A preferred Env plasmid pBR322dtVSVG encodes the Vesicular Stomatitis Virus Envelope Glycoprotein (VSVG) and has the sequence shown in SEQ ID No. 4 (see 8th for the plasmid card).

All Plasmids, except the transfer plasmid, preferably contain a known polyadenylation signal (polyadenylation site), preferably bovine growth hormone.

Around the risk of heterologous recombination with naturally occurring Minimizing viruses or the transfer plasmid are the for the functional proteins (Gag, Pol, Vif, Rev and Tat) of the visnavirus coding genes used in the invention, in comparison to the wild type sequences that naturally in the Visnavirus, changed. The genes are preferably of synthetic, non-naturally occurring nucleic acid sequences built up. Compared to the naturally occurring genes are the genes at the nucleic acid level so changed, that they are as strong as possible at the nucleic acid level differ, but still for encode the same proteins. At the same time, the DNA sequences for translation optimized in human cells.

These change is achieved by using codons other than those of the wild type virus genes. The codons are substituted through codons that are still for the same amino acid residues stand, but deviate in as many bases as possible. Of the possible alternative codons are selected the most frequently is used in human cells and is different from that used in the wild type Codon is different. This change preferably results in a homology between the synthetic Genes and the wild-type which is lower than 65%, preferably 61%. The change of the invention of on average every third nucleotide leads advantageously to the destruction of the Cis-acting inhibitory sequences and also the destruction of the Packing signal in gag coding area.

The Homology between the transfer plasmid and the VTR, Gag and Pol Plasmids are further reduced since most of the gag coding Area and the complete pole-coding Area was removed from the transfer plasmid. Through this strategy The frequency of recombinations is advantageously over a hundred times reduced.

The changed Genes are preferably of synthetic oligonucleotides with lengths between 40 to 80 nucleotides assembled by PCR and sequential Ligations (ligations) and enzymatic digestions (digestion) composed are. This procedure has three advantages: First, the Assembling smaller fragments while creating the derivatives, a greater variety of restriction sites. Second, that allows Omitting a single fragment from the completed plasmid in a simple way, the proportion of the transfer plasmid, that of the virus is derived, reduce. Third, the design was based of the virus on a consensus sequence instead of a characterized Wild-type virus. This design strategy allows accuracy better control of the sequence than a RT-PCR of a wild-type virus.

The Gene, which for Gag polyprotein encodes preferably has a nucleic acid sequence according to SEQ_ID No. 11th

The Gene, which for the Pol polyprotein encodes, preferably has a nucleic acid sequence according to SEQ_ID No. 12th

The Genes, which for Vif, Rev and Tat encode preferably nucleic acid sequences according to SEQ_ID No. 13 to 15.

• a.) ein Gag-Plasmid enthaltend eine künstliche Nukleinsäuresequenz codierend für VLV-Gag, bevorzugt gemäß SEQ_ID No. 11 und bevorzugt unter der Kontrolle des CMV-Promotors, und ein Polyadenylierungssignal, bevorzugt vom bovinen Wachstumshormon, wobei dieses Plasmid kein kompetentes Verpackungssignal enthält;
• b.) ein Transferplasmid – enthaltend eine VLV-Nukleinsäuresequenz beinhaltend Cis-acting Elemente (cis-acting sequence elements) benötigt für die reverse Tanskription des Plasmidgenoms, wobei besagter Vektor enthält: (i) den Gag-Leader (Gag-L) und inaktivierte Sequenzen von gag p16 (D-p16), beinhaltend ein kompetentes Verpackungssignal (competent packaging signal) und die Cis-acting Elemente D-tat, D-rev und D-env, welche nicht für funktionale Proteine codieren, da die Start-Codons entfernt sind, und ein kompetentes Rev-response element (RRE); (ii) eine Ribozymkassette enthaltend entweder eine Leitsequenz (guide sequence) mit einer Länge von 10 bis 20 Nukleotiden oder eine Füllsequenz (stuffer) flankiert von Restriktionsstellen, bevorzugt Psrl Restriktionsstellen, die katalytische Domain eines Gruppe I Introns, bevorzugt ein Tetrahymena 26S rDNA Intron, und entweder eine Nukleinsäure codierend für das korrekte Gegenstück der zu reparierenden Target-RNA, welches auf die geschnittene Target-RNA gespleißt wird oder eine Klonierungsstelle flankiert von Restriktionsstellen, bevorzugt Bael-Restriktionsstellen, wobei die Ribozymkassette bevorzugt unter der Kontrolle des SV40-Promotors ist;
• c.) ein Pol-Plasmid enthaltend eine künstliche Nukleinsäuresequenz codierend für VLV-Pol, bevorzugt gemäß SEQ_ID No. 12 und bevorzugt unter der Kontrolle des SV40-Promotors, und ein Polyadenylierungssignal, bevorzugt vom bovinen Wachstumshormon, wobei dieses Plasmid kein kompetentes Verpackungssignal enthält;
• d.) ein VTR-Plasmid enthaltend künstliche Nukleinsäuresequenzen codierend für VLV-Vif, Rev und Tat, bevorzugt gemäß SEQ_ID No. 13 bis 15 und bevorzugt unter der Kontrolle des SV40-Promotors, und ein Polyadenylierungssignal, bevorzugt vom bovinen Wachstumshormon, wobei dieses Plasmid kein kompetentes Verpackungssignal enthält;
• e.) ein Env-Plasmid enthaltend eine künstliche Nukleinsäuresequenz codierend für ein (i) virales Hüllprotein, welches nicht vom Visnavirus ist, bevorzugt das Vesicular Stomatitis Virus Envelope Glykoprotein (VSVG) und ein Polyadenylierungssignal, bevorzugt vom bovinen Wachstumshormon, wobei dieses Plasmid kein kompetentes Verpackungssignal enthält.

In a preferred embodiment of the invention, the recombinant lentiviral gene delivery system comprises:

• a.) A gag plasmid containing an artificial nucleic acid sequence coding for VLV gag, preferably according to SEQ ID no. 11 and preferably under the control of the CMV promoter, and a polyadenylation signal, preferably of bovine growth hormone, which plasmid does not contain a competent packaging signal;
• b.) a transfer plasmid containing a VLV nucleic acid sequence comprising cis-acting sequence elements required for reverse transcription of the plasmid genome, said vector containing (i) the gag leader (Gag-L) and inactivated Sequences of gag p16 (D-p16) containing a competent packaging signal and the cis-acting elements D-tat, D-rev and D-env which do not encode functional proteins since the start codons are removed are, and a competent Rev-response element (RRE); (ii) a ribozyme cassette containing either a guide sequence with a length of 10 to 20 nucleotides or a filling sequence (stuffer) flanked by restriction sites, preferably Psrl restriction sites, the catalytic domain of a group I intron, preferably a Tetrahymena 26S rDNA intron, and either a nucleic acid encoding the correct counterpart of the target RNA to be repaired which is spliced onto the cut target RNA or a cloning site flanked by restriction sites, preferably Bael restriction sites, wherein the ribozyme cassette is preferably under the control of the SV40 promoter;
• c.) a Pol plasmid containing an artificial nucleic acid sequence coding for VLV pol, preferably according to SEQ ID no. 12 and preferably under the control of the SV40 promoter, and a polyadenylation signal, preferably of bovine growth hormone, which plasmid does not contain a competent packaging signal;
• d.) a VTR plasmid containing artificial nucleic acid sequences coding for VLV-Vif, Rev and Tat, preferably according to SEQ ID no. 13 to 15 and preferably under the control of the SV40 promoter, and a polyadenylation signal, preferably of bovine growth hormone, which plasmid does not contain a competent packaging signal;
• e.) an env plasmid containing an artificial nucleic acid sequence encoding a viral envelope protein other than visnavirus, preferably the vesicular stomatitis virus envelope glycoprotein (VSVG) and a polyadenylation signal, preferably bovine growth hormone, which plasmid is not a competent Packaging signal contains.

One Particularly preferred Gag plasmid is pBr322dtVLVgag with the nucleic acid sequence according to SEQ_ID No. 1. Contains this plasmid the for Gag coding sequence according to SEQ_ID No. 11th

One Particularly preferred Pol plasmid is pBr322dtVLVpol with the nucleic acid sequence according to SEQ_ID No. 2. Contains this plasmid the for Pol coding sequence according to SEQ ID No. 12th

One A particularly preferred VTR plasmid is pBr322dtVLV-VTR, having the nucleic acid sequence according to SEQ_ID No. 3. Contains this plasmid the for Vif, Rev and Tat coding sequences according to SEQ ID no. 13 to 15.

• a.) das Gag-Plasmid pBr322dtVLVgag mit der Nukleinsäuresequenz gemäß SEQ_ID No. 1,
• b.) ein Transferplasmid mit einer Nukleinsäuresequenz ausgewählt aus pBR322dtVLVt-βGal, pBR322dtVLVdAPP, pBR322dtVLV-ECAP, pBR322dtVLV-PLAP, pBR322dtVLV-GFP, pBR322dtVLV-PsrBae mit den Nukleinsäuresequenzen gemäß SED_ID No 5 bis 10,
• c.) das Pol-Plasmid pBr322dtVLVpol mit der Nukleinsäuresequenz gemäß to SEQ_ID No. 2,
• d.) das VTR-Plasmid pBr322dtVLV-VTR mit der Nukleinsäuresequenz gemäß SEQ_ID No. 3,
• e.) das Env-Plasmid pBr322dtVSVG mit der Nukleinsäuresequenz gemäß SEQ_ID No. 4.

In a particularly preferred embodiment of the invention, the recombinant lentiviral gene transfer system contains:

• a.) the gag plasmid pBr322dtVLVgag with the nucleic acid sequence according to SEQ ID no. 1,
• b.) a transfer plasmid having a nucleic acid sequence selected from pBR322dtVLVt-βGal, pBR322dtVLVdAPP, pBR322dtVLV-ECAP, pBR322dtVLV-PLAP, pBR322dtVLV-GFP, pBR322dtVLV-PsrBae having the nucleic acid sequences according to SED_ID No 5 to 10,
• c.) the pol plasmid pBr322dtVLVpol with the nucleic acid sequence according to SEQ ID NO. 2,
• d.) the VTR plasmid pBr322dtVLV-VTR with the nucleic acid sequence according to SEQ ID no. 3,
• e.) the Env plasmid pBr322dtVSVG with the nucleic acid sequence according to SEQ ID no. 4th

To transduction of the recombinant leintiviral gene transfer system in cells, the cells become replication-deficient lentivirus particles to produce.

One second component of the invention is a process for the preparation of replication-deficient visnavirus particles, including the Transfection of a packaging cell line (producer cells) with the above-described gene transfer system according to the invention

The Transfection of packaging cell lines (producer cells) is with Standard methods of molecular biology, eg. Calcium phosphate coprecipitation, carried out.

preferred Cells for virus production is cell lines 293 (ATCC # CRL-1573) or 293T (ATCC # CRL-11268) or 293ts / A1609 (DuBridge et al., 1987, Mol Cell Biol. 7: 379-387).

Around to produce a lentiviral stock, cultured the packaging cell line (producer cells) under cell culture conditions, which are sufficient to limit the production of replication-deficient lentiviral To allow particles in the cell line. The replication-deficient Lentivirus particles are then released from the packaging cells (producer cells).

The Visnavirus particles obtained by a method according to the invention and their use for the ribozyme-mediated Repair of a mutant gene sequence in eukanotic cells are also Subject of the present invention.

• a.) Klonierung eines heterologen Gens oder eines Genabschnittes, welcher für das wiederherzustellende korrekte Gegenstück (z. B. die Wildtyp-cDNA-Sequenz der mutierten Target-RNA) codiert und welches dem Abschnitt der mutierten Target-RNA entspricht, welcher ausgetauscht werden muss, in die Klonierungsstelle des Transferplasmids,
• b.) Transfektion einer Verpackungszelllinie (producer cells) mit dem so erhaltenen Transferplasmid und den weiteren Plasmiden des erfindungsgemäßen Gentransfersystems;
• c.) Wachstum der Verpackungszellen (producer cells) unter Zellkulturbedingungen, die ausreichend sind, um die Produktion von replikationsdefizienten lentiviralen Partikeln in der Zelle zu erlauben, und Sammeln der replikations-defizienten Lentivirus-Partikel von den Verpackungszellen (producer cells).
• d.) Infektion von eukanotischen Zellen mit den replikations-defizienten Visnavirus-Partikeln.

A further component of the present invention is also a method for the ribozyme-mediated repair of a mutant target RNA in eukarotischen cells, comprising the steps:

• a.) Cloning of a heterologous gene or of a gene segment which is to be restored correct counterpart (eg the wild-type cDNA sequence of the mutated target RNA) and which corresponds to the portion of the mutated target RNA which needs to be exchanged into the cloning site of the transfer plasmid,
• b.) transfection of a packaging cell line (producer cells) with the transfer plasmid thus obtained and the other plasmids of the gene transfer system according to the invention;
• c.) growth of the producer cells under cell culture conditions sufficient to allow the production of replication-deficient lentiviral particles in the cell, and collecting the replication-deficient lentivirus particles from the producer cells.
• d.) Infection of eukanotic cells with the replication-deficient visnavirus particles.

In front the infection of eukanotic cells become the replication-deficient Visna virus particles sterilized and cell debris removed, preferably by filtration through filters with a pore size of 0.22 microns. Prefers the visnavirus particles are subsequently concentrated and macromolecules are removed, preferably with 0.1 μm Filter.

For the infection the replication-deficient Visnavirus particles are preferred resuspended in phosphate buffered saline.

By the infection of eukaryotic cells either in cell culture or by administration of the replication-deficient visnavirus particles By means of in vivo injection, the ribozyme is introduced into the cell and initiated the ribozyme-mediated repair.

The Efficiency of infection and ribozyme-mediated repair can be be controlled by placing a marker gene (such as GFP) in the transfer plasmid added becomes. In many cases However, the presence of a marker gene is not desirable, in particular in therapeutic approaches ribozyme-mediated repair. In these cases, therefore, the efficiency of infection and ribozyme-mediated repair by specific Measurement of repaired RNA detected. This is preferably achieved by extracting mRNA from the infected tissue or cell culture and RT-PCR is performed becomes specific for primers the 5 'target RNA and the heterologous nucleic acid, the 3 'on the cut Spliced target RNA has been. receives one RT-PCR DNA product, this demonstrates a successful ribozyme-mediated Repair.

The The following examples are intended to illustrate the invention and are intended to do so To enable a person skilled in the art to practice the invention. she are intended to cover the scope of the invention as defined in the claims is, not limit.

1 shows a plasmid map of the plasmid pBR322Lnk1, a starting plasmid for the transfer plasmids of the lentiviral gene transfer construct expression system.

pBR322Lnk1 was removed from the cloning plasmid pBR322 by removal of the tetracycline resistance gene (tet) between the Styl and Hindlll restriction sites created. A synthetic linker (Lnk1) was used instead of the removed Sequence inserted. This linker contains Lnk1 a swallows blunt-end restriction site into which a PCR product can be cloned. Flank two aarl restriction sites the Swal recognition and restriction site. The Aarl recognition agencies are placed so that the associated restriction sites on the ends of the DNA fragment that clones into the Swal site fall becomes. Subsequent digestion of the derivative by ligation is formed in the Swal site releases a DNA fragment which from the inserted Sequence is derived and in which the four base pairs at the ends converted into 4 lawn pair-long 5 'overhangs are.

This Sticky-end DNA fragment is used to plasmids of the present Establish invention.

The plasmid pBR322Lnk1 further contains the sequences of the original pBR322 which are required for the replication, transcription and translation of the plasmid: two promoters (P1 P, P3 P), Shine-Dalgarno acting as ribosomal binding site (RBS) ribosomes Sequences (SD SEQ), the ampicillin-resistance gene (β-lactamase) AP ^r , an L-strand and an H-strand Y-effector site (LY EFF and NY EFF), an origin of replication (ORI) and a for the gene encoding ROP protein.

To obtain pBR322Lnk1, the cloning plasmid pBR322 (ATCC 37017) is digested with the restriction enzymes Hindill (New England Biolabs) and Styl (New England Biolabs). To carry out the double digestion, 1 μg pBR322 DNA, 1.5 μl Styl and 1 μl HindIII and 1 μl BSA are added to 50 NEB buffer 3 in 500 μl reaction tube added. The mixture is incubated in a thermomixer (Eppendorf, Wesseling-Berzdorf, Germany) at 37 ° C. and 300 rpm for 1 hour. This digestion cuts out the tetracycline resistance gene. Digestion yields two fragments: the 3021-bp Styl-HindIII fragment (1370-29) and the 1340-bp HindIII-Styl fragment (30-1369). These fragments are separated by electrophoresis in agarose. The larger DNA fragment is excised from the electrophoresis gel, the DNA extracted and divided into three aliquots.

Around to obtain the linker Lnk1, the oligonucleotide Lnk1 s (agcttcacctgcatttaaatgcaggtgc) with another oligonucleotide (Lnk1rc; caaggcacctgcatttaaatgcaggtga) anneals and phosphonates (5 'with the T4 polynucleotide kinase; NEB, M0201). Lnk1 is inserted into the pBR322 Styl-HindIII fragment ligated. The resulting plasmid is called pBR322Lnk1. pBR322Lnk1 is transformed into E. coli, the plasmid is expanded overnight and then extracted.

2 shows a plasmid map of pBR322dtVLVt-βGal, an example of the VLV-based transfer plasmid of the invention, which splices the transcript of the reporter gene E. coli β-galactosidase gene 3 'into a target mRNA.

pBR322dtVLVt-βGal contains one Ribozyme cassette labeled with "ribozyme" and a filling sequence (stuffer) denoted by "ssrl". The filling sequence "sPsrl" can be replaced by a Ribozyme guide sequence (guide-sequence), which recognizes a specified target mRNA, be replaced. The coding sequence spliced by the ribozyme 3 ', is this for the E. coli β-galactosidase (βGal) or odierende Reporter gene "LacZ", in which the Codon usage has been altered to optimal human shape. The ribozyme cassette is under the control of the SV40 promoter. The ribozyme corresponds the catalytic domain of the 26S rDNA intron of T. thermophylia (GenBank V01416).

Around the reverse transcription, packaging and integration of the To enable genomes contains that Transfer plasmid cisacting elements of the VLV, those in the nucleic acid sequences designated "Gag-L", "pbs", "D-env", "D-rev," D-tat "," D-p16 ". These sequences do not code for functional proteins, since the start codons (each for the first Encode methionine) are removed.

Of the Gag leader "Gag-L" and the inactivated p16 sequence "D-p16" also contain one Competent packaging signal (competent packaging signal).

The Transfer plasmid pBR322dtVLVt-βGal contains a rev-response element (RRE), responsible for the export is the virion mRNA from the nucleus of the host cell, and one as Shine-Dalgarno sequence (SD SE) acting on ribosome binding site (RBS) binding parts.

The RRE is for the export of viral RNA from the nucleus of the packaging cell (producer cell) needed and acts by interacting with Rev. REV2-L is the nucleic acid sequence, which contains the RRE. empirical Results show that the sequence rev 2 is necessary for the function the GTC is. U5 and R5 are necessary elements of the 5'-LTR, while U3 is the untranslated leader sequence of the 3 'LTR. The nucleic acid sequence of pBR322dtVLVt-βGal is in SEQ_ID no. 9 listed.

Around pBR322dtVLVt-βGal to obtain the plasmid pBR322Lnk1 with the restriction enzyme SwaI (New England Biolabs) digested. Through this digestion you get one linear blunt-end plasmid. The digested plasmid is washed with alkaline Calf intestinal phosphatase (Calf Intestinal Alkaline Phosphatase - CIP, New England Biolabs) dephoshorylated and purified on an agarose gel.

The ribozyme cassette-encoding DNA and the cis-acting elements are assembled from synthetic oligonucleotides which are assembled into four DNA fragments: U5-p16, tat-RRE, R2L-LacZ and U3-R3. A scheme for the construction of the plasmid pBR322dtVLVt-βGal is given in 3 shown. In summary, pBR322dtVLVt-βGal is obtained by assembling the fragments U5-p16, tat-RRE, R2L-LacZ and U3-R3 to the fragment "VLV-LacZ" and cloning "VLV-LacZ" into the digested pBR322Lnk1.

The U5 p16 fragment contains the primer binding site (pbs), the VLV long terminal repeats R5 and U5, the gag leader (Gag-L, SEQ ID NO 16) and the inactivated p16 region of gag ("D-p16", SEQ ID NO. 17). The tat-RRE fragment contains the inactivated sequences "D-env" (SEQ ID NO: 20), "D-rev" (SEQ ID NO: 19), "D-tat" (SEQ ID NO: 18), and Sequences RRE (SEQ ID NO 21), Rev2-L and rev2. The R2L-LacZ fragment contains the ribozyme cassette, including the PsRI filling sequence (sPsRI), the SV40 promoter and the gene encoding βGal (LacZ). The U3-R3 fragment contains the U3 'and R3 portions of the VLV, including the 3' LTR.

These DNA fragments U5-p16, tat-RRE, R2L-LacZ and U3-R3 were made from DNA fragments assembled with 44 to 73 base pairs, which in turn by means of PCR of pairs of chemically synthesized single-stranded DNA oligonucleotides (Sigma-Genosys Ltd., Cambridge CB2 4EF, UK). at In the PCR, the oligonucleotides acted both as primers and as templates. The 3'-oligonucleotide corresponded in length half of the fragment plus 12 bases. The 5 'oligonucleotide corresponded to 4 bases of the 3'-following fragment, plus half said fragment plus 10 bases. The PCR extends the Oligonucleotides, both sense and antisense, to the full length of the DNA fragment. Each DNA fragment, which has been configured overlaps with the following DNA fragment around 4 bases. The overlap was designed so that the following ligation the fragments too the complete Combine plasmid.

PCR Thermocycler-Bedingungen:

The PCR is performed in a sterile, nuclease-free 0.5 ml PCR tube in a thermocycler (MultiCycler PTC 200, Biozym Diagnostics GmbH, Hessisch Oldendorf, Germany), which was preheated to 95 ° C under the following conditions: PCR- reaction:

PCR thermocycler conditions:

The PCR yields fragments with lengths of 67 to 123 bp (depending on the length of the oligonucleotides used in the PCR), which are extracted from the reaction mixture by agarose gel electrophoresis on a 2% gel (61-3112 Certified Low-Malt agarose, Bio-Rad Laboratories GmbH, Munich, Germany) in a Sub Cell Model 192 Cell exactly according to the manufacturer's instructions (Sub-Cell ^® Model 96 and Model 192 Agarose Gel Electrophoresis Systems Instruction Manual, Bio-rad) are separated.

The band containing the DNA fragment is excised and the DNA extracted from the gel according to the manufacturer's instructions (QIAquick PCR purification extraction kit, Qiagen, Hilden, Germany). The DNA fragment is inserted into pBR322Lnk1 by blunt-end ligation into the Swal site. The plasmid thus obtained is transformed into competent E. coli exactly according to the manufacturer's instructions transformed (One Shot ^® TOP10 Chemically Competent E. coli Invitrogen GmbH, Karlsruhe, Germany). The plasmid is expanded overnight and extracted with a mini-prep kit (Qiagen).

All other PCRs, plasmid duplications (plasmid expansions) and extractions are carried out the same way, though not mentioned otherwise.

The Plasmid DNA is mixed with BfuAl (New England Biolabs GmbH, Frankfurt on the Main, Germany) for 2 h at 37 ° C digested. The fragment corresponding to the ligated PCR product, was purified from an agarose gel after electrophoresis.

The DNA fragments U5-p16, tat-RRE, R2L-LacZ and U3-R3 are added to this By several consecutive rounds of cutting with assembled with the restriction enzyme BfuAl and religation.

Of the SV40 early promoter is generated from the plasmid pcDNA3.1 / CT-GFP-TOPO (Invitrogen) by PCR amplification of for the promoter coding sequence with the primers CTGTGGAATGTGTGTCAGTTAGGGTGT and CTATTGGTTTAAAGACTAGCTACCAGGTGCAT having the PCR conditions described above.

Around pBR322dtVLVt-βGal to obtain the DNA fragments U5-p16, tat-RRE, R2L-LacZ and U3-R3, the SV40 early promoter ligated into pBR322Lnk1 into the Swal site

4 shows a plasmid map of pBR322dtVLVdAPP, a second example of the VLV-based transfer plasmid of the present invention. The plasmid pBR322dtVLVdAPP is a transfer plasmid designed to repair mutant βAPP.

The Ribozyme cassette contains here a guide sequence "GS" (guide sequence), which complementary to the target sequence in the βAPP mRNA and the βAPP pre-mRNA is, the catalytic domain of the 26S rDNA intron ribozyme of Tetrahymena thermophylia and a Sequence for the homologous unmutated sequence of the 3 'region of APP (nucleotides 1168 to 2266).

The Leading sequence "GS" recognizes the targets βAPP mRNA and the βAPP pre-mRNA. The Ribozyme is capable of the sequence passing through the 3 'region the ribozyme cassette is coded (and for the homologous, non-mutated Sequence encoded) to splice onto the cut Traget RNA.

The nucleic acid sequence of pBR322dtVLVdAPP is shown in SEQ_ID no. 6 listed.

The production of pBR322dtVLVdAPP proceeds like that of pBR322dtVLVt-βGal (s. 2 ), except that the coding sequence for the 3 'region of APP is inserted instead of that of LacZ and the leader sequence "GS" instead of the filling sequence sPSRI.

The Leading sequence (GS), which for the ribozyme is used, which is the PC stimulated by both NGF 12 and HEK293 Cells Expressed Amyloid Precursor Protein (APP) is GGTCCTCGGTCGGCAGCA. This GS serves as a recognition and splice site for the Ribozyme. The GS DNA fragment is made up of two oligonucleotides: GCTCGGTCCTCGGTCGGCAGCA and TAATTGCTGCCGACCGAGGACT. By annealing These two oligonucleotides are obtained a DNA fragment, which for the encoded anti-APP ribozymal leader (GS) that is compatible with the 5'-overhang of the ribozyme and the 3 'overhang of the SV40 promoter. By altering this GS sequence can the ribozyme can be adapted to almost any target RNA.

5 shows a plasmid map of pBR322dtVLVgag, an example of a Gag plasmid encoding the polyprotein gag of the visna lentivirus (VLV).

The Plasmid pBR322dtVLVgag contains an expression cassette for the production of the polyprotein VLV-gag (which contains the proteins p16, p25 and p14). The for VLV gag coding sequence is completely artificial. The codon usage the for VLV gag coding sequence was coding towards the VLV gag Wild-type sequence altered to a maximum, to minimize the risk of homologous recombination with the wild-type. additionally became the codon usage for optimized translation in human cells and for VLV gag coding sequence with a bovine polyadenylation signal Growth hormone (BGH pA) equipped. Expression of VLV gag coding sequence is under the control of cytomegalovirus (CMV) promoter.

The nucleic acid sequence of pBR322dtVLVgag is in SEQ_ID no. 1 listed.

pBR322dtVLVgag was constructed based on pBR322Lnk1 and by assembly of the VLV gag coding sequence starting from synthetic oligonucleotides and by successive ligations ments and restriction digests in the same way as under 2 prepared for the transfer plasmid described.

The cytomegalovirus immediate-early gene (CMV) promoter is generated from the plasmid pcDNA3.1 (+) (Invitrogen, Cat # V790-20) by PCR amplification of the promoter coding sequence with the primers:
GTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAG and
GAGCTCTGCTTATATAGACCTCCCACCG
obtained under the PCR conditions described above.

6 Figure 4 shows a plasmid map of pBR322dtVLVpol, an example of a Pol plasmid encoding the polyprotein Pol of Visna lentivirus (VLV).

The Plasmid pBR322dtVLVpol contains an expression cassette for the production of the polyprotein VLV-Pol. The VLV Pol coding sequence is completely artificial. The codon usage of the VLV-Pol coding sequence was opposite the wild-type VLV pol gene was altered to the maximum risk to minimize homologous recombination with the wild-type. In addition will the codon usage for optimized translation in human cells and for VLV pol coding sequence with a polyadenylation signal of bovine growth hormone (BGH pA). The expression of the VLV-Pol coding sequence is under the control of the Simian Virus 40 (SV40) promoter.

The nucleic acid sequence of pBR322dtVLVpol is in SEQ_ID no. 2 listed.

pBR322dtVLVpol was constructed based on pBR322Lnk1 and by assembly of the VLV-Pol coding sequence starting from synthetic oligonucleotides and by successive ligation and restriction digestions in the same way as described below 2 prepared for the transfer plasmid described.

7 Figure 4 shows a plasmid map of pBR322dtVLV-VTR, an example of the VTR plasmid containing nucleic acid sequences encoding Vif, Rev and Tat accessory VLV proteins.

The Plasmid pBR322dtVLV-VTR contains an expression cassette for the production of VLV proteins Vif, Tat and Rev. The codon usage the for Vif, Tat and Rev coding sequences deviate maximally from wild-type to reduce the risk of homologous recombination with the wild type to minimize. additionally is the codon usage for optimized translation in human cells and that for VLV-Vif, Tat and Rev coding sequence with a polyadenylation signal of bovine growth hormone (BGH pA). The expression the for VLV-Vif, Tat and Rev coding sequence is under control Simian Virus 40 (SV40) promoter.

The nucleic acid sequence of pBR322dtVLV-VTR is shown in SEQ_ID no. 3 listed.

pBR322dtVLV-VTR was constructed based on pBR322Lnk1 and by assembling the Vif, Tat and Rev coding sequence starting from synthetic oligonucleotides and sequential ligation and restriction digests in the same manner as described below 2 prepared for the transfer plasmid described.

8th shows a plasmid map of pBR322dtVSVG, an example of the Env plasmid encoding a viral envelope protein (envelope protein Env), which is not from the Visnavirus.

The Plasmid pBR322dtVSVG contains an expression cassette for the Vesicular Stomatitis Virus Envelope Glycoprotein (VSVG). The for VSVG coding sequence was for the human codon usage is optimized and comes with a polyadenylation signal of bovine growth hormone (BGH pA). The expression the for VSVG coding sequence is under the control of the CMV promoter.

The Plasmid pBR322dtVSVG was constructed based on pBR322Lnk1 and by assembly constructed of oligonucleotides.

The nucleic acid sequence of pBR322dtVSVG is shown in SEQ_ID no. 4 listed.

9 shows a plasmid map of pBR322dtVLV-ECAP, a third example of the erfindungsge permeable transfer plasmid which splices the transcript of the reporter gene E. coli alkaline phosphatase 3 'into a target mRNA.

pBR322dtVLV-ECAP contains a ribozyme cassette similar to that of (in 2 shown) plasmid pBR322dtVLVt-βGal resembles. In this ribozyme cassette, the filling sequence sPsrl can be replaced by a ribozymal guide-sequence GS for a specified target mRNA. The coding sequence spliced 3 'by the ribozyme here is the sequence coding for the E. coli reporter gene alkaline phosphatase, in which the codon usage was changed to the optimal human form.

The nucleic acid sequence of pBR322dtVLV-ECAP is shown in SEQ. ID. 7 listed.

The production of pBR322dtVLV-ECAP is identical to that of pBR322dtVLVt-βGal (s. 2 ) with the difference that the DNA fragment LacZ is replaced by the sequence coding for the E. coli alkaline phosphatase optimized for human codon usage, which is constructed from synthetic oligonucleotides as described above.

10 shows a plasmid map of pBR322dtVLV-PLAP, a fourth example of the transfer plasmid according to the invention, which splices the transcript of the reporter gene placental alkaline phosphatase (PLAP) 3 'into a target mRNA.

The plasmid pBR322dtVLV-PLAP contains a ribozyme cassette similar to that of (in 2 shown) plasmid pBR322dtVLVt-βGal resembles. In this ribozyme cassette, the filling sequence sPsrl can be replaced by a ribozymal guide sequence (GS) for a specific target mRNA. The coding sequence spliced 3 'by the ribozyme is the reporter gene coding for placental alkaline phosphatase (PLAP) in which the codon usage has been changed to the optimal human form.

The nucleic acid sequence pBR322dtVLV-PLAP is shown in SEQ. ID. 8 listed.

The production of pBR322dtVLV-PLAP is similar to that of pBR322dtVLVt-βGal (s. 2 ) with the difference that the DNA fragment LacZ is replaced by the placental alkaline phosphatase (PLAP) coding sequence obtained as a synthetic human codon usage optimized sequence and as described above from synthetic Oligonucleotides is constructed.

11 shows a plasmid map of pBR322dtVLV-GFP, a fifth example of the transfer plasmid according to the invention, which splices the transcript of the reporter gene Enhanced Green Fluorescent Protein (eGFP) 3 'into a target mRNA.

The plasmid pBR322dtVLV-GFP contains a ribozyme cassette similar to that of the (in 2 shown) plasmid pBR322dtVLVt-βGal resembles. In this ribozyme cassette, the filling sequence sPsrl can be replaced by a ribozymal guide sequence (GS) for a specific target mRNA. The coding sequence spliced 3 'by the ribozyme is here the reporter gene Enhanced Green Fluorescent Protein (eGFP), in which the codon usage has been changed to the optimal human form.

The nucleic acid sequence of pBR322dtVLV-GFP is shown in SEQ. ID. 10 listed.

The production of pBR322dtVLV-GFP proceeds in the same way as that of pBR322dtVLVt-βGal (s. 2 ) with the difference that the DNA fragment LacZ is replaced by the reporter gene sequence coding for Enhanced Green Fluorescent Protein (eGFP), which is derived from the plasmid pEGFP (BD Biosciences Clontech, Heidelberg, Germany) by a PCR amplification of the promotor coding sequence is obtained with the primers gtgagcaagggcgaggagctg and ttacttgtacagctcgtccatgccg.

12 Figure 4 shows a plasmid map of pBR322dtVLV-PsrBae, a sixth example of the transfer plasmid of the invention, which contains a cloning site into which a sequence 3 'to be spliced by the ribozyme can be inserted.

pBR322dtVLV-PsrBae contains a ribozyme cassette similar to that of (in 2 shown) plasmid pBR322dtVLVt-βGal resembles. In this ribozyme cassette, the filling sequence sPsrl can be replaced by a ribozymal guide-sequence (GS) for a specific target mRNA. pBR322dtVLV-PsrBae further contains a cloning site (csBael) flanked by Bael restriction sites into which a sequence to be 3 'spliced by the ribozyme can be inserted after Bael restriction digestion of the plasmid.

The nucleic acid sequence pBR322dtVLV-PsrBae is in SEQ_ID no. 5 listed.

The production of pBR322dtVLV-GFP proceeds in the same way as that of pBR322dtVLVt-βGal (s. 2 ), with the difference that the DNA fragment LacZ is replaced by the cloning site csBae, which is constructed by the insertion of a linker instead of the βGal coding sequence.

Production of replication-deficient Virion Vektoroartikeln:

Around to obtain replication-deficient virion vector particles Cells with an example of the recombinant lentiviral according to the invention Gene transfer system transfected: the gag plasmid pBR322dtVLVgag, the transfer plasmid pBR322dtVLV-GFP, the pol plasmid pBR322dtVLVpol, for the accessory proteins Vif, Rev and Tat encoding VTR plasmid pBR322dtVLV-VTR, and that for the Env plasmid pBR322dtVSVG encoding VSV envelope protein.

In order for the ribozyme cassette to target the mRNA encoding the β-amyloid precursor protein (APP), the filling sequence (stuffer) sPsrl in the transfer plasmid pBR322dtVLV-GFP here becomes the leader sequence GGTGGCGCTCCTCTGGGG, which encodes the mRNA coding for APP recognizes (s. 4 replaced. The ribozyme cassette will splice the EGFP coding sequence to the APP mRNA.

The Cell line 293T (ATCC CRL-11268) is supplemented with Dulbecco's modified Eagle's medium 10% fetal calf serum (fetal calf serum - FCS), Penicillin, streptomycin and glutamine (Invitrogen) are maintained. The 293T cells were analyzed by the calcium phosphate coprecipitation method, as described (Soneoka et al., 1995, Nucleic Acids Res. 23: 628-633). The supernatant the transfected 293T cells that replication-deficient Contains virion vector particles, is through a filter with 0.45 microns Given pore size and in 500 μl Aliquots stored at -80 ° C. The frozen aliquots containing the replication-deficient virion vector particles contain a lentiviral stock (lentiviral stock) The following is for the infection of cells is needed.

Infection of cells with Virion vector particles:

The Infection of cells with virion vector particles is in the absence and in the presence of aphidicolin performed to determine whether or not non-dividing cells can be transfected.

Virion vector particles are prepared as described above.

Cultured rat pheochromocytoma cells PC12 (ATCC CRL-1721) and HEK293 cells are placed in 96-well plates at a density of 2 x 10 ⁴ cells / well. PC12 cells are adjusted in 82.5% Ham's F12K medium with 2 mmol / L L-glutamine to 1.5 g / L sodium bicarbonate, 15% horse serum, 2.5% fetal calf serum (Invitrogen) and 50ng / ml 2.6s Neuronal growth factor (nerve growth factor, Sigma-Aldrich Chemie, Deisenhofen, Germany). HEK293 cells are seeded in 90% Minimum Essential Medium (Eagle) with 2 mmol / L L-glutamine and Earle's BSS adjusted to 1.5 g / L sodium bicarbonate, 0.1 mmol / L nonessential amino acids and 1.0 mmol / l Sodium pyruvate, and 10% heat-inactivated horse serum (Invitrogen) cultivated.

To One day the medium is removed and the cells are kept for 2 to 4 hours with serial dilutions (1:30, 1: 100, 1: 300, 1: 1000, 1: 3000, 1: 10,000) of the virion vector particle preparations in a total volume of 150 μl / well in a 24-well plate or 30-50 μl / well in a 96-well plate incubated. Fresh medium is added. The Number of GFP-positive cells is determined 2 days after infection.

To arrest PC12 and HEK293 cells in the G ₁ / S phase of the cell cycle, the cells are placed in a 96-well plate at a density of 4 x 10 ⁴ to 5 x 10 ⁴ cells / well, using aphidicolin at a concentration of 5 μg / ml (Sigma) throughout the whole culture period.

The number of transfected cells is determined by determining the GFP fluorescence in lysates of three independent transfections - in the presence of aphidicolin at 5 μg / ml (+) or its absence (-) (see 13 ).

The transfected PC12 and HEK293 cells are suspended in Luciferase Cell Culture Lysis Reagent (Promega, Mannheim, Germany) and the intensity of the GFP fluorescence of the lysates in a 1420 Multi-Label Counter (Victor, Wallac, Turku, Finland) the method of Schnell et al. (2000, Development of a soap-inactivating, minimal lentivirus vector based on simian immunodeficiency virus, Human Gene Therapy 11: 439-447). In 13 the mean values and standard deviations of GFP fluorescence measurements are shown (GFU, Green Fluorescence-forming Units).

In Presence and absence of Aphidicholin will be a large number of PC12 and HEK293 cells infected with virion vector particles. Although the titers lower when treated with aphidicholine were, the results show significant that non-dividing Cells efficiently with the replication-deficient invention Virion vector particles can be infected.

Repair of a point mutation by ribozyme-mediated RNA repair:

When an example of ribozyme-mediated repair with the lentiviral according to the invention Gene transfer system will repair the point mutations in the for the β-amyloid precursor protein coding messenger RNA described.

A number of point mutations in the gene encoding β-amyloid precursor protein (APP, GenBank D87675) elicit clinically significant pathologies (Online Mendelian Inheritance in Man * 104760, NCBI, Bethesda, USA). Of these, the correction is accessible by an exchange (substitution) of a 3 'subsequence:

The transfer plasmid used to transfer the self-splicing ribozyme which corrects for these point mutations is pBR322dtVLVdAPP represented by the plasmid map in FIG 4 ,

Around replication-defective virion vector particles are obtained Cells as described above with the transfer plasmid pBR322dtVLVdAPP, the gag plasmid pBR322dtVLVgag, the pol plasmid pBR322dtVLVpol, for the accessory proteins Vif, Rev and Tat encoding VTR plasmid pBR322dtVLV-VTR, and that for the VSV envelope Protein encoding Env plasmid pBR322dtVSVG transfected.

PC12 cells and HEK293 cells are infected with virion vector particles as described above. Prior to infection, the PC12 cells are stimulated for seven days with 50 ng / ml neuronal growth factor (NGF) (N6009, Sigma-Aldrich Chemie GmbH, Germany).

alternative can primary Cells infected in cell culture or the virion vector parficles by in vivo injection be applied.

Around To control the efficiency of infection, mRNA is taken from the infected cell culture (or infected tissue) with the Oligotex Direct mRNA Micro Kit (Qiagen, Hilden, Germany) accordingly extracted from the manufacturer's instructions.

A RT-PCR with primers specific for the 5 'substrate RNA and the heterologous nucleotide sequence spliced 3 'onto the substrate RNA, is running. The RT-PCR is carried out according to the method of Bangsow, Huch, Male and miller (2002, Chapter 5.2.2.1 Schrimpf (Ed) in Genetic engineering methods, Spektrum Akademischer Verlag GmbH, Heidelberg, Germany) with the conditions described above for the PCR. When Antisense priming is called GACGATCACTGTCGCTATGACAACAC and as a sense primer TGCCGACCGAGGAC-TAATGTCCCAGGTCATGAGAGA used.

The DNA fragments obtained by PCR are subjected to agarose gel electrophoresis separated. The agarose gel electrophoresis is performed as described above. Anti-sense priming is specific for wild-type βAPP. The Presence of a band of RT-PCR product (length 952bp) indicates the successful trans-splicing of the trans-induced ribozyme out. The production of an RT-PCR DNA product demonstrates the success of the mRNA repair.

3'APP

Bereich der für das β-Amyloid-precursor protein (APP) codierenden Sequenz, der 3' gespleißt wird, um eine mutante APP mRNA zu reparieren

Aarl

Erkennungsstelle für das Restriktionsenzym Aarl

Apr

Ampicillin Resistenzgen (β-Lactamase)

ATCC

American Type Culture Collection

Bael

Bael-Restriktionsstelle

csBael

Klonierungsstelle (cloning site – cs) flankiert von Bael-Restriktionsstellen für den Einbau einer Target-Sequenz

BGH

bovines Wachstumshormon (Bovine growth hormone)

CMV

Cytomegalievirus

D-env

Inaktivierte VLV-Envelope-Nukleidsequenz

D-p16

Inaktivierte VLV-p16-Nukleidsequenz

D-rev

Inaktivierte VLV-rev-Nukleidsequenz

D-tat

Inaktivierte VLV-tat-Nukleidsequenz

ECAP

Humanisierte Sequenz codierend für die E. coli Alkalische Phosphatase

EGFP

Enhanced Green Fluorescent Protein

EGFP-PA

EGFP-Polyadenylierungssignal

Gag

Humanisierte für Gag-codierende Sequenz

Gag-L

Gag-Leader-Sequenz

GFP

Green Fluorescent Protein

GFU

grüne Fluoreszenz-bildende Einheiten (Green Fluorescence-forming Units)

GS

Ribozym-Leitsequenz (guide-sequence)

H Y EFF

H-strand Y-eftector site

HEK293

Human embryonic kidney fibroblast cell-line

HindIII

Erkennungsstelle des Restriktionsenzyms HindII

L Y EFF

L-strand Y-Effector-Site

LacZ

Sequenz codierend für eine humanisierte Form der E. coli β-Galaktosidase

Lnk1

Linker 1

LTR3

3' Long Terminal Repeat

LTR5

5' Long Terminal Repeat

ORI

Bakterieller Startpunkt für die Replikation (Origin of replication)

p

Plasmid

P10'

P10-Loop der katalytischen Sequenz des Ribozyms

P1P

Bakterieller Promotor P1

P3P

Promotor P3

PA

Polyadenylierungssignal (Polyadenylation signal)

pbs

Primerbindungsstelle (Primer binding site)

PC12

Rat pheochromocytoma cell line

PLAP

Optimierte Sequenz codierend für humane plazentare alkalische Phosphatase (placental alkaline phosphatase)

Pol

Humanisierte Sequenz codierend für das VLV Pol-Polyprotein

sPsrl

Füllsequenz (stuffer) flankiert von Psrl Restriktionsstellen für den Einbau einer (Leistsequenz guide sequence)

Psrl

Erkennungsstelle des Restriktionsenzyms Psrl

R5

5'-Repeat

Rev

2 Zweites rev-Exon

Rev2-L

Rev 2-Leader

Ribozyme

Katalytische Ribozym-Sequenz

ROP

Rop-Protein codierendes Gen

RRE

Rev Response Element

SD SEQ

Shine-Dalgarno-Sequenz

Styl

Erkennungsstelle des Restriktionsenzyms Styl

SV40

Simianvirus 40 Early Promoter

Swal

Erkennungsstelle des Restriktionsenzyms Swal

Tat

Humanisierte Sequenz codierend für den Transactivator of transcription

U3

Nichttranslatierte 3'-Sequenz (Untranslated 3'-Sequence)

U5

Nichttranslatierter 5'-Bereich des VLV (Untranslated 5'-Region of VLV)

vif

Humanisierte Sequenz codierend für den Viral-infectivity factor

VLV

Visna-Lentivirus

VSVG

Vesicular Stomatitis Virus Envelope Glykoprotein codierende Sequenz

The following abbreviations are used in the description of the invention and in the figures:

3'APP

Portion of the β-amyloid precursor protein (APP) coding sequence spliced 3 'to repair a mutant APP mRNA

Aarl

Recognition site for the restriction enzyme Aarl

April

Ampicillin resistance gene (β-lactamase)

ATCC

American Type Culture Collection

Bael

Bael restriction site

csBael

Cloning site (cs) flanked by Bael restriction sites for incorporation of a target sequence

BGH

bovine growth hormone (bovine growth hormone)

CMV

cytomegalovirus

D-env

Inactivated VLV Envelope Nukleidsequenz

D-p16

Inactivated VLV p16 Nukleidsequenz

D-rev

Inactivated VLV-rev Nukleidsequenz

D-tat

Inactivated VLV Tat Nukleidsequenz

ECAP

Humanized sequence encoding E. coli alkaline phosphatase

EGFP

Enhanced Green Fluorescent Protein

EGFP-PA

EGFP polyadenylation signal

gag

Humanized for gag coding sequence

Gag-L

Gag leader sequence

GFP

Green Fluorescent Protein

GFU

green fluorescence forming units

GS

Ribozyme guide sequence

HY EFF

H-beach Y-eftector site

HEK293

Human embryonic kidney fibroblast cell-line

Hind

Recognition site of the restriction enzyme HindII

LY EFF

L-strand Y Effector Site

LacZ

Sequence coding for a humanized form of E. coli β-galactosidase

Lnk1

Linker 1

LTR3

3 'Long Terminal Repeat

LTR5

5 'Long Terminal Repeat

ORI

Bacterial starting point for replication (Origin of replication)

p

plasmid

P10 '

P10 loop of the catalytic sequence of the ribozyme

P1P

Bacterial promoter P1

P3P

Promoter P3

PA

Polyadenylation signal (polyadenylation signal)

pbs

Primer binding site

PC12

Rat pheochromocytoma cell line

PLAP

Optimized sequence encoding human placental alkaline phosphatase

pole

Humanized sequence encoding the VLV Pol polyprotein

sPsrl

Filler sequence (stuffer) flanked by Psrl restriction sites for insertion of a (power sequence guide sequence)

Psrl

Recognition site of the restriction enzyme Psrl

R5

5'-Repeat

Rev

2 Second rev-exon

Rev2-L

Rev 2 Leader

ribozymes

Catalytic ribozyme sequence

ROP

Gene encoding rop protein

RRE

Rev Response Element

SD SEQ

Shine-Dalgarno sequence

Styl

Recognition site of the restriction enzyme Styl

SV40

Simian Virus 40 Early Promoter

Swa

Recognition site of the restriction enzyme Swal

did

Humanized sequence encoding the transactivator of transcription

U3

Untranslated 3 'sequence (untranslated 3' sequence)

U5

Untranslated 5 'region of the VLV (Untranslated 5' region of VLV)

vif

Humanized sequence coding for the viral infectivity factor

VLV

Visna lentivirus

VSV

Vesicular stomatitis virus envelope glycoprotein coding sequence

Claims (15)

Recombinant lentiviral gene transfer system for ribozyme-mediated Repair of a target RNA containing: a. a first plasmid containing a for the polyprotein gag of the visna lentivirus (VLV) encoding nucleic acid sequence, wherein said plasmid does not encode other VLV proteins and does not contain a competent packaging signal, b. a second plasmid as transfer plasmid containing: (i) VLV nucleic acid sequences, which for the reverse transcription of the plasmid genome required cis-acting sequence elements contain (cis-acting sequence elements), (ii) a competent Packaging signal and (iii) a ribozyme cassette containing the sequence of the catalytic domain of a self-splicing intron ribozyme (Group I intron) and a heterologous gene or a section of a Gene, which for the accurate counterpart the target RNA to be repaired, or a cloning site, into which a heterologous gene or part of a gene is responsible for the accurate counterpart the target RNA to be repaired can be used, c. a third plasmid containing a coding for the VLV polyprotein Pol Nucleic acid sequence the plasmid is not for encodes other VLV proteins and no competent packaging signal contains d. a fourth plasmid containing for the VV, Rev and Tat VLV proteins encoding nucleic acid sequences, wherein the plasmid is not for encodes other VLV proteins and no competent packaging signal contains e. a fifth Plasmid containing a nucleic acid sequence coding for a viral envelope protein (envelope protein - Env), which is not derived from Visnavirus and wherein the plasmid not for VLV proteins coded and contains no competent packaging signal. Recombinant lentiviral gene transfer system according to claim 1, characterized in that for VLV Gag, Pol, Vif, Rev and Tat coding sequences of synthetic, not in nature occurring nucleotide sequences are constructed. Recombinant lentiviral gene transfer system according to claim 2, characterized in that the sequences responsible for VLV gag, Pol, Vif, Rev and Tat encode the sequences according to SEQ_ID. No. 11 to 15 include. Recombinant lentiviral gene transfer system according to one of claims 1 to 3, characterized in that the viral envelope protein Env encoded by the fifth plasmid is selected from the group Ve Systolic Stomatitis Virus Envelope Glycoprotein (VSVG), Amphotropic MuLV Envelope (4070A Env), Ebola Envelope, gp64 Envelope Glycoprotein of Bakulovirus, Influenza Virus A Haemagglutinin (HA), Jaagsiekte Sheep Retrovirus (JSRV) Envelope, Lymphocytic Choriomeningitis Virus (LCMV) Glycoprotein ( LCMV-GP), Mokola Virus Envelope Glycoprotein (MK-G), Murine Leukemia Virus Envelope, Rabies Virus Envelope Glycoprotein and RD114 Retrovirus Envelope (RD114-Env). Recombinant lentiviral gene transfer system according to a the claims 1 to 4, characterized in that the self-splicing intron Ribozyme (group I intron) is a Tetrahymena 26S rDNA intron. Recombinant lentiviral gene transfer system according to claim 5, characterized in that the Tetrahymena 26S rDNA intron selected is from the organisms Tetrahymena hyperangularis, Tetrahymena sonneborni, Tetrahymena cosmopolitanis, Tetrahymena pigmentosa and Tetrahymena thermophylia. Recombinant lentiviral gene transfer system according to a the claims 1 to 6, characterized in that the ribozyme cassette additionally a guide sequence complementary to a region of the target RNA or one flanked by restriction sites stuffer (stuffer sequence) contains. Recombinant lentiviral gene transfer system according to a the claims 1 to 7, characterized in that coding for the VLV protein Gag nucleic acid sequence under the control of the cytomegalovirus (CMV) promoter and the for the VLV proteins Pol, Vif, Rev and Tat and the envelope protein Env coding nucleic acid sequences under the control of Simian Virus 40 (SV40) promoter. Recombinant lentiviral gene transfer system according to a the claims 1 to 8 containing the plasmids with the sequences according to SEQ_ID No. 1 to 4 and a transfer plasmid selected from the sequences according to SEQ ID No. 5 to 10. The plasmids of the recombinant lentiviral gene transfer system for ribozyme-mediated Repair of a Target RNA with the Sequences according to SEQ_ID. No. 1 to 10. The nucleic acid sequences coding for the VLV proteins Gag, Pol, Vif, Rev and Tat of the recombinant lentiviral Gene transfer system for Ribozyme-mediated repair of a target RNA containing the synthetic, non-naturally occurring nucleotide sequences selected from SEQ_ID no. 11 to 15. Process for the preparation of replication-deficient Virion vector particles comprising the transfection of packaging cells (producer cells) with a recombinant lentiviral gene transfer system according to one of the claims 1 to 9, where the cells are replication-deficient virion particles to produce. The method of claim 12, wherein the packaging cells selected are from cell lines 293 or 293T or 293ts / A1609. Replication-deficient virion vector particles were obtained according to the method of claim 12 or 13. Procedure for Ribozyme-mediated repair of a target RNA in eukaryotic Cells, comprising the steps: a.) Klovierung a heterologous Gene or a gene section, which for the correct counterpart of repairing target RNA encoded in the transfer plasmid of the lentiviral Gene transfer system according to one of claims 1 to 9, b.) Transfection of packaging cells (producer cells) with the transfer plasmid thus prepared and the other plasmids of the lentiviral gene transfer system according to any one of claims 1 to 9 c.) growth of the packaging cells under cell culture conditions, which are sufficient to make the production of replication-deficient allow lentiviral particles in the cell, and collect the replication-deficient Visna virion particles from the packaging cells, d.) Infection of eukaryotic cells with the replication-deficient Visna virion particles.