JPH11511018A - Self-deleting vectors for gene therapy - Google Patents

Abstract

(57) SUMMARY The present invention involves the development of vectors for somatic cell gene therapy. This vector introduces a complete transcription unit containing the promoter, protein coding sequence and polyadenylation sequence into the genome of a mammalian cell. Once integrated, the vector removes most viral and non-viral sequences unrelated to the transcriptional unit, thus suppressing gene expression by transcriptional silencing, recruiting endogenous retroviruses, activating oncogenes or immunizing. Problems encountered with conventional retroviral vectors, such as the appearance of a response, can be avoided. The present invention relates to (i) the natural life history of retroviruses, including the replication of terminal control sequences U5 and U3 to create long terminal repeats (LTRs), and (ii) two specificities of the mammalian genome. The ability of site-specific recombinases to excise any sequences located between the target sequences. That is, the retrovirus of the present invention introduces into the genome a site-specific recombinase coding sequence and at least one recombinase-specific target sequence along with a transcription unit that expresses a therapeutic gene.

Description

DETAILED DESCRIPTION OF THE INVENTION Self-deleting vectors for gene therapy Field of the invention The present invention relates to a specially made vector that introduces a transcription unit into a eukaryotic cell, preferably such that upon integration all sequences unrelated to the transcription unit are removed. Only one transcription unit, including any natural or synthetic promoter / enhancer sequences, protein coding sequences and polyadenylation sites, is retained in the genome. By removing itself from the genome, the vectors of the present invention allow transcriptional disruption by the introduced gene or a gene adjacent to the integration site, activation of cell oncogenes, recruitment of endogenous retroviruses, and emergence of an immune response. Such problems encountered with conventional vectors (eg, retroviruses and their vectors) can be avoided. Background of the Invention Retroviruses are RNA viruses that replicate via a DNA intermediate. The end of the viral RNA genome is flanked by short sequence repeats (R) and unique sequences (U5 and U3) that control DNA synthesis, integration, transcription, and RNA processing. Between the control regions are the coding sequences for the major structural proteins of the virus particle (gag and env) and the enzymes present in the particle (pol, protease, reverse transcriptase and integrase) (FIG. 1). . Shortly after infection, viral RNA is converted to DNA by reverse transcriptase. The process is initiated by the cellular tRNA acting as an extension primer and binding to a complementary region in the viral genome. This region, also called the primer binding site (PBS), is located immediately downstream of U5 and is essential for virus replication. Prior to integration, the terminal sequences of the viral genome are replicated such that long terminal repeats (LTRs), each containing the U3, R and U5 regions, flank the retroviral genome. This type of linear DNA molecule is integrated into the genome (FIG. 1). The LTR sequence is maintained in the integrated virus, also called the provirus, but two nucleotides (nt) are missing from each end. The cellular DNA sequence does not change, but upon integration, the provirus replicates 4-6 nt, with 4-6 nt repeats flanking the ends. As a provirus, the retroviral genome is replicated along with cellular DNA and transcribed by RNA polymerase II as a cellular gene. Proviral transcription is controlled by a promoter / enhancer sequence present in the U3 region of the 5 'LTR. Polyadenylation transcripts begin at the junction of U3 and R (cap site) in the 5 'LTR and end at the R of the 3' LTR, which contains the signal for polyadenylation. Full-length (genomic) RNA is transported from the nucleus to the cytoplasm, packaged into viral particles and budding or translated from cells to produce gag and pol proteins. Some of the RNA is spliced to produce mRNA encoding env. It is possible to modify a retrovirus to introduce a gene into the mammalian genome. If some of the regulatory sequences in the LTR remain unchanged, the retroviral genome can be deleted without impairing its ability to replicate in cells that express proteins necessary for reverse transcription, integration and particle formation. For this, the vector DNA is transfected into a cell line containing the complete retroviral genome or a helper virus. Helper virus is made such that particles do not assemble due to small deletions containing the sequence (Y) between U5 and gag. Because the vector DNA does not contain a Y deletion, the recombinant transcript is packaged and eliminated from the cell as a viral particle. In addition to Y, a gag sequence can also enhance the ability of the vector to be packaged. To date, retroviral vectors have been the most efficient means of introducing foreign genes into mammalian cells. Thus, retroviruses are used in over 80% of all approved gene therapy trials. However, several factors have hindered the practical use of traditional retroviruses as gene therapy vectors. First, the introduced gene is often inactivated by methylation or binding of a transcriptional repressor to the viral genome (1-3). These repressors have been shown to bind to the primer binding sites of some retroviruses, and their simple removal would interfere with virus replication. Second, retroviruses integrate almost randomly into the genome, so integration often results in mutations, which enhance expression of adjacent genes (4,5). It has been described that activation of the adjacent proto-oncogene is followed by malignant transformation of infected cells (6). The activation mechanism involves transcriptional enhancement by the upstream U3 promoter or a nearby U3 enhancer. Third, viral vector sequences, such as package signals and leader sequences, can recombine with endogenous retroviruses to generate new undesirable types of infectious viruses (7-9). Finally, viral or non-viral sequences in certain vectors can provoke an immune response that eliminates the introduced cells (10). A review of gene therapy is given in (26). It is an object of the present invention to provide new vectors that do not exhibit the disadvantages of the prior art vectors. This object is achieved by a vector system useful for gene therapy comprising at least one coding sequence for a site-specific recombinase and at least one target sequence specifically recognized by this recombinase. The vector system may be a DNA or RNA vector. Currently, there are several vectors that can be used for gene therapy and are suitable as starting materials for the production of the vector system of the present invention. The site-specific recombinase may be any recombinase that specifically recognizes a target sequence. The prior art provides some examples of site-specific recombinases and corresponding target sequences. One of skill in the art can select the location of the sequence encoding the recombinase and the target sequence in the vector, depending on which portions of the vector are removed after integration into the mammalian genome. The site-specific recombinase may optionally be encoded by another vector, which is simultaneously contained in the cell having the vector containing the target sequence and further the sequences necessary for integration of the vector into the genome. Further, the recombinase may be added as a protein to cells containing the vector having the target sequence. In a preferred embodiment, the combination of recombinase and target sequence used is recombinase Cre and target sequence loxP. In a further embodiment, the recombinase is Flp and the target sequence is frt. The vector of the present invention may contain an arbitrary transcription unit, which is a transcription unit composed of a gene encoding a desired function to be introduced into a mammalian genome. Often this gene encodes a protein that is not properly prepared by the cell receiving the vector of the invention. In a further preferred embodiment, the vectors of the invention are retroviral vectors, most preferably retroviral DNA. If a retroviral vector is used, the target sequence is preferably inserted into the U3 and / or U5 region, which may further contain a transcription unit. In a further preferred embodiment, the vectors of the present invention may include a viral promoter and / or enhancer. The introduction of the vector of the invention into a mammalian cell results in a cell containing, after removal of the undesired vector portion of the genome, at least one target sequence introduced by the vector. In many cases, the vector will also contain additional transcription units, which are contained within the mammalian cell genome. When the transcription unit contains a gene that is normally also contained in a mammalian cell but does not function for some reason, the gene introduced into the cell via the vector of the present invention may be one that is naturally present in the cell. It will have a different chromosomal environment as compared to the gene. When a vector sequence (eg, the proviral genome) is deleted from the chromosome, typically a few nucleotides of the proviral genome remain in the genome of the cell. Thus, as used herein, "essentially no provirus genome" means that preferably less than 600 base pairs, more preferably less than 100 base pairs, of nucleotides remain in the genome. By using the vector of the present invention, any DNA can be incorporated into the genome of a mammalian cell. The location of the target sequence in the vector allows to predetermine which parts of the vector will be deleted from the genome after vector integration. The present invention involves the development of novel vectors, particularly retroviruses, for introducing transcription units into the genome of eukaryotic cells for therapeutic or non-therapeutic purposes. This vector has a site-specific recombination system that includes a recombinase such as Cre or Flp and at least one specific target sequence such as loxP or frt. When this system is integrated, it is activated to remove all viral and non-viral sequences unrelated to the transcription unit. This type of retrovirus can preferably avoid all the unwanted side effects encountered with conventional retroviral vectors. Preferred retroviral vectors of the invention contain a transcription unit, preferably in the U3 or U5 region, including a promoter, a protein coding sequence, and a polyadenylation sequence. Also preferably in the U3 or U5 region, the retrovirus of the invention contains at least one synthetic or natural target sequence of a site-specific recombination system. In such a system, DNA fragments flanked by target sequences having the same orientation are removed by the corresponding recombinase (FIG. 2). The retrovirus of the present invention replicates by inserting a target sequence into the U3 or U5 region during replication. This places most of the viral genome within the same target sequence and allows the recombinase to remove the proviral mass. Only one copy of the introduced transcription unit containing the gene of interest remains in the genome. This recombinase expression is achieved in trans by introducing a protein or a plasmid expressing the recombinase into the introduced cells, or preferably in cis by expressing the recombinase from the provirus itself (FIG. 3). , 4). In this case, the sequence encoding the recombinase is preferably located between the target sequences outside the proviral control region (LTR). Their expression is preferably controlled by a second synthetic or natural promoter. The polyadenylation signal is preferably provided by the R region of the 3 'LTR. If the recombinase cassette is in the reverse orientation with respect to the provirus, transcription ends with a hidden proviral polyadenylation signal or with additional synthetic or natural polyadenylation sequences cloned into the virus in the reverse orientation. . Preferably, the retrovirus of the present invention is free of enhancers and / or promoters. However, the retroviruses of the present invention continue to contain their own promoter and enhancer sequences. The retrovirus of the present invention is preferably used to introduce a therapeutic gene into mammalian cells, but can also be used for basic research. The retroviruses of the present invention may also introduce potentially harmful sequences as long as they are deleted at the appropriate time after integration. For example, a potential application is a vector based on human retrovirus (HIV, HTLVI). The invention includes a mammalian cell containing at least one site-specific recombination target (eg, loxP or frt) and at least one protein coding sequence introduced by a retrovirus of the invention. These cells are mostly deficient in retroviral sequences. Finally, the present invention provides a method for introducing a cDNA sequence into a mammalian genome (eg, site-specific recombinases associated with transfection of producer cells, infection of mammalian cells, and deletion of a recombinase expression cassette with a retrovirus of the present invention). Expression). BRIEF DESCRIPTION OF THE FIGURES FIG. 1 is a schematic representation of the genome of a prior art retrovirus. FIG. 2 is a schematic diagram of a site-specific recombination system. Figures 3 and 4 are schematic representations of the retrovirus of the present invention, P = promoter; Ts = site-specific target sequence; PCS = protein coding sequence; Rec = recombinase. FIG. 5 is a schematic diagram of the retrovirus vectors U3pgklxtkneo and U3pgklxtkneoMCCre. FIG. 6 is a Southern blot analysis of clones expressing U3pgklxtkneo before and after transfecting MCCre. In the first six lanes, the DNA was digested with the enzymes NdeI and XbaI, which do not cut in the provirus. Lanes 7-12 contain HindIII digested DNA from Cre expressing (+) and non-expressing (-) clones. FIG. 7 shows Southern blot analysis with DNA obtained from clones expressing U3pgklxtkneo (A) and U3pgklxtkneoMCCre (B). Note the reduced signal intensity of certain HindIII fragments from the Cre expression clone (B). Variable bands correspond to the number of proviruses. FIG. 8 is a schematic diagram of the retroviral vectors pggSVCreU3lxpgkpuro and pggSVCreU3lxSVpuro. FIG. 9 shows the recombination of U31xpgkpuroSVCre provirus in NIH3T3 cells. (A) Predicted structure of provirus before and after site-specific recombination. (B) Southern blot analysis of U31xpgkpuroSVCre provirus. Genomic DNA was cut with EcoRI, fractionated on agarose gel, blotted on nylon filter, ³² Hybridized to a P-labeled pgk-probe. Lanes 1-14, clones 1-14 expressing U3lxpgkpuroSVCre. The ubiquitous 7 kb constant band is the endogenous pgk promoter. (C) PCR analysis of clones expressing U31xpgkpuroSVCre. Genomic DNA was amplified with Cre- (top) or b-actin- (bottom) specific primers. Amplification products were separated on a 1% agarose gel and visualized by staining the lane with ethidium bromide as follows: M, molecular weight standard (1 kb BRL ladder); 1-14, clone 1 expressing U3xpgkpuroSVCre. -14,15, a clone expressing a single copy of U3pgklxtkneo (negative control); 16, a clone expressing a single copy of U3pgklxtkneoMCCre (positive control). FIG. 10 shows recombination of U31xSVpuroSVCre provirus in NIH3T3 cells. (A) Predicted structure of provirus before and after site-specific recombination. (B) Southern blot analysis of U31xSVpuroSVCre provirus. Cellular DNA was cut with EcoRI (left) or HindIII (right) and treated as described in the legend of FIG. ³² Hybridized to P-labeled SV40-probe. Lanes 1-12, clones 1-12 expressing U3lxSVpuroSVCre. (C) PCR analysis of clones expressing U3xSVpuroSVCre. Genomic DNA was amplified with Cre- (left) or b-actin- (right) specific primers. Amplification products were separated on a 1% agarose gel and visualized by staining the lane with ethidium bromide as follows: M, molecular weight standard (1 kb BRL ladder); 1-12, clone 1 expressing U3xSppurSVCre. 12; 13-14, clones expressing a single copy of U3pgkltkneo (positive control); 15-16, clones expressing a single copy of U3pgklxtkneo MCC re (negative control). The term "vector system" refers to at least one vector capable of introducing a recombinase coding sequence and a target sequence for the recombinase into the genome of a cell. The sequence encoding the recombinase and the target sequence may be located on two different vectors, but are preferably present in one vector. The recombinase may be optionally introduced into the cell as a protein, such that the vector system need not include the coding sequence for the recombinase. The term "retrovirus" refers to any RNA virus that replicates via a DNA intermediate. Some such viruses require the presence of another virus, such as a helper virus, for passage. That is, a retrovirus is intended to mean one having a substantial deletion or mutation in its RNA. The term "control region" refers to the region of the retrovirus that replicates after infection and before integration. Control regions include U3 and U5 regions. Such regions also include LTR regions. The term "transcription unit" refers to a sequence of nucleic acids that includes a natural or synthetic promoter, protein coding sequence, and a polyadenylation signal. The promoter may contain an enhancer. The term "protein coding sequence" refers to a nucleotide sequence that encodes a polypeptide chain that has therapeutic value or that in some way interferes with cellular metabolism. It also includes a polypeptide, commonly referred to as a "selectable marker", that distinguishes cells that express the polypeptide chain from cells that do not express the polypeptide chain. The term "target sequence" or "site-specific target sequence" refers to a synthetic or natural nucleotide sequence recognized by a site-specific recombinase. Examples of such sequences include loxP (11-13) from P1 phage or frt (14-16) from Saccharomyces cerevisiae. The term "recombinase" or "site-specific recombinase" refers to a synthetic, natural, or recombinant enzyme that binds, cleaves, recombines with a specific target sequence. Examples of such are Cre recombinase from P1 phage (13) or Flp recombinase from Saccharomyces cerevisiae (14). The present invention relates to self-deleting vectors, preferably retroviruses, which are preferably used to introduce therapeutically valuable genes into somatic cells. FIG. 5 shows two preferred embodiments of the present invention. In the above vector -pgggU3pgglkxtkneo-, a transcription unit consisting of the mouse phosphoglycerate kinase (pgk) promoter (17), the loxP target sequence (11), and the thymidine kinase / neomycin phosphotransferase (tkneo) fusion gene was replaced with an enhancer-free gene. It was inserted into the 3'-U3 region of the MoMuLV retroviral vector. In the lower vector, an additional transcription unit encoding MCCre (where Cre = Cre recombinase, MC = HSV thymidine kinase promoter fused to polyomalousie T enhancer) was inserted between the LTRs of the viral body. Viral and LTR-mediated replication places the MCCre between the loxP site along with other viral and non-viral sequences, and Cre recombinase causes the integrated protease other than one copy of pgk-lx-tkneo containing the LTR. Allows you to remove most of the virus. FIG. 8 is two additional preferred embodiments of the present invention. In both constructs, the MC promoter was replaced with the simian virus 40 (SV40) promoter and the tkneo fusion gene was replaced with a puromycin resistance gene. In the above vector-pgSVCreU31xpgkpuro-, the puromycin resistance gene is controlled by the pgk promoter. In the lower vector-pgSVCreU 31 x SVpuro-the same U3 gene is controlled by the SV40 promoter. In these examples, the loxP site in the 3 'LTR was located upstream of the transcription unit. In the preferred embodiment described above, the transcription unit and the site-specific recombinase target are within U3, but this is not required. Both the transcription unit and the target sequence may be in the U5 region or other parts of the vector, depending on which part of the vector is deleted. Finally, Cre recombination may be provided in trans as a native protein or as a plasmid expressing the recombinase. Example 1 To determine if the site-specific recombination target could delete adjacent proviral sequences after integration, we inserted the transcription unit -pgk-loxP-tkneo- into the U3 region of the enhancer-free Moloney murine leukemia virus. (Fig. 5). Plasmid Cre recombinase and loxP sequences were obtained from pMCCre and PGEM 30, respectively (11). The mouse phosphoglycerate-kinase-promoter (pgk) was obtained from ppgkCat (18) and the SV40 / puromycin-acetyltransferase cassette was obtained from pBABEpuro (19). The tk neo gene was obtained by joining the EheI / SpeI PCR amplification product of the HSV thymidine kinase coding sequence (20) to the SpeI / NheI amplification product of the neomycin-phosphotransferase coding sequence (21). In frame fusions were made by deleting the thymidine kinase stop codon and Neo-AUG. The pgklxtkneo cassette was cloned blunt-ended into the unique NheI site of pggU3en (-) to obtain pggU3pgkl xtkneo. pggU3en (-) was obtained by removing neo from pggU3Neoen (-) (21) and subcloning the viral sequence as an SstI fragment into the backbone of pBABEpuro. pgklxtkneo corresponds to the site of loxP as the PstI / EcoRI fragment of pGEM30, pgk as the XbaI / BglII fragment of pgkCAT, and tkneo as the blunt-end fragment, and the site of the pBluescriptIIKS polylinker (Stratagene). By doing so, it was assembled in pBluescript IIKS. To obtain pggU3pgklxtkneoMCCre, the PCR amplification product of MCCre was cloned into the unique XhoI site of pggU3pgklxtkneo. Cells and viruses NIH3T3 cells and BOSC23 cells (22) were grown in DMEM (Gibco) medium supplemented with 10% fetal calf serum (Gibco). As described by Pear et al. (22), transient transfection of BOSC23 cells resulted in a recombinant retrovirus free of helper virus. 10 ^Five NIH3T3 cells were infected by incubating with the filtered virus supernatant for 24 hours in the presence of 4 μg / ml polybrene (Aldrich). Clones expressing the provirus were isolated by selection in media containing 2 μg / ml puromycin (Sigma) or 1 mg / ml G418 (Gibco) for 7 days. Southern blot hybridization As already described (21) ³² DNA hybridization was performed using a P-labeled probe. Southern blots were scanned with a PhosphoImager (Molecular Dynamics) and analyzed with ImageQuantNT software (Molecular Dynamics). result Previous studies have demonstrated that the U3 region of MoMuLV can tolerate relatively large amounts of external sequence (up to 5 kb, (23)). These are replicated with the viral sequence to create a long terminal repeat (LTR) flanked by integrated proviruses (reviewed in (24)). Thus, after insertion of the site-specific recombination-loxP-target sequence into the 3'-U3 region of the retroviral vector, LTR-mediated replication places the viral genome between the two loxP sites, thus It was predicted that this would be susceptible to cleavage by Cre-recombinase. To examine this prediction, a transcription unit consisting of the mouse phosphoglycerate kinase (pgk) promoter (17), the loxP target sequence (11), and the thymidine kinase / neomycin phosphotransferase (tkneo) fusion gene was replaced with an enhancer-free gene. It was inserted into the 3'-U3 region (pgU3en (-), (21)) of the MoMuLV retrovirus vector to obtain pggU3pgklx tkneo- (FIG. 5). This plasmid was transfected into BOSC23 helper cells (22) to obtain a recombinant virus, which was used to infect NIH3T3 cells. LTR-mediated replication should produce a pgklxtkneo flanked provirus in infected cells (FIG. 6). To test this, after selection in G418, the resulting 12 neomycin resistant clones were analyzed by Southern blot. LTR-mediated replication was confirmed in each case using a restriction endonuclease that cleaves the LTR (data not shown). To determine whether Cre recombinase deleted the sequence flanked by loxP and fused the 5 'pgk promoter to the 3' tkneo gene (FIG. 6), two cell lines expressing U3pgklx tkneo were isolated from Cre2, respectively. Co-transfected with the expression plasmids MCCre (11) encoding recombinase and puromycin resistance and pBABEpuro (19). Genomic DNA extracted from the cell pool obtained by puromycin selection was digested with HindIII (an endonuclease that cleaves the LTR) (FIG. 6). When hybridized to the neo-probe, the non-recombinant provirus produces a constant 4.7 kb band that can accommodate the sequence flanked by loxP (Figure 6, lanes 7-12). This band is very weak or completely abolished in the clones expressing Cre (FIG. 6, lanes 8, 9, 11, 12), indicating that most proviruses have undergone recombination. Was. That is, the loxP site inserted into U3 allows Cre recombinase to excise most proviruses other than one LTR. The portion of the proviral DNA remaining in the chromosome after deletion of most proviral DNA can be easily tested by sequence analysis of the chromosomal insertion site and sequence comparison with the vector used to introduce the foreign gene. it can. Example 2 To develop a vector that has all the necessary components for self-deletion into mammalian cells, we propose that the loxP site inserted into U3 will allow recombination when Cre is expressed from the same provirus. I checked whether I would receive it. The coding sequence of Cre controlled by the MC promoter was inserted into pggU3pgklxtkneo between LTRs to obtain pggU3pgklxtkneoMCCre. To obtain infectious virus, pggU3pgklxtkneoMCCre was transfected into BOSC23 cells as described in Example 1. Neomycin resistant clones obtained after infecting NIH3T3 cells and selecting in G418 were analyzed by Southern blot as described in Example 1. The provirus obtained from this vector was expected to cleave itself after integration. Genomic DNA from several independent clones expressing U3pgklxtkneo and U3pgklxtkneoMCCre was digested with HindIII (a restriction endonuclease that cuts both proviruses downstream of tkneo). When hybridized to neo, the non-recombinant provirus retains all sequences flanked by loxP, resulting in constant bands of 4.7 kb and 6 kb, respectively. This band was still present in the Cre-expressing clone, but it was 5-10 fold weaker than in the non-expressing clone (FIG. 7, B-lane). Since both types of clones contained comparable numbers of proviruses, the results indicate that a significant number of U3pgklxtkneoMCCre proviruses had deleted sequences flanked by loxP. Example 3 To improve the efficiency of self-deletion of the Cre-expressing provirus, two additional constructs were made using an additional promoter and a selectable marker gene. Plasmid The loxP fragment of pGEM30 containing the Cre region of MCCre, the SV40 promoter of pBABEpuro, and a HindIII site downstream was inserted into the BamHI, XhoI and NheI sites of pGgU3en (-), respectively, as blunt-ended fragments. The blunt-end pgk-puro- or SV40-puro expression cassette was ligated to the HindIII site of loxP to obtain pggSVCreU 31 × pgkpuro and pgSVCreU31 × SVpuro. Cells and viruses Same as described in Examples 1 and 2. DNA hybridization and PCR As already described (21) ³² DNA hybridization was performed using a P-labeled probe. Southern blots were scanned with a PhosphoImager (Molecular Dynamics) and analyzed with ImageQuantNT software (Molecular Dynamics). For the PCR assay, 150 ng of genomic DNA was subjected to 40 cycles (94 ° C for 30 seconds, 60 ° C for 1 second) using Cre specific primers 5'-TTAGCTAGCATGCCCAAAGAAGAAGAAG-3 'and 5'-GGAGCTAGCCTAATCGCCAT CTTCCAG-3'. Minutes, 72 ° C. for 1 minute). Control PCR was performed under the same conditions except using actin primers as previously described (25). result Infectious virus was obtained by transfecting the vectors -pgSVCreU31xpgkpuro- and -pgSVCre U31xSVpuro- (FIG. 8) into BOSC23 cells. The recovered virus was used for infection of NIH3T3 cells, and puromycin-resistant clones were isolated after 7 days of selection. Genomic DNA of the puromycin-resistant clones obtained from either construct was analyzed by Southern blot. To identify recombination of the U31xpgkpuroSVCre provirus, the DNA was digested with EcoRI, an endonuclease that cuts both proviruses before each promoter (FIGS. 9A, 10A). When hybridized to pgk, the non-recombinant provirus retains all sequences flanked by loxP, resulting in a constant band of 3.1 kb (FIG. 9B, lanes 5, 9, 12-14). . This band should disappear from the Cre expressing clone (FIG. 9A). Thus, this band disappeared from 9 of the 14 U31xpgkpuroSVCre expressing clones, indicating that most proviruses have been recombined (FIG. 9B, lanes 1-4, 6-8, 10). Additional bands of various sizes are fragments extending from the EcoRI site of the 3 ′ LTR to the site of adjacent cellular DNA (FIG. 9B). However, all clones, including those recombined on Southern blots, produced Cre-specific amplification products when analyzed by PCR (FIG. 9C). Although the recombined clone produced 10-50 fold less amplification product (FIG. 9C), the results suggest that the recombinase does not achieve the threshold levels required for recombination uniformly. Significantly higher recombination efficiencies were obtained with U31xpgkpuroSVCre. As shown in FIG. 10B, none of the clones produced a 3 kb restriction fragment when hybridized to SV40, and only variable bands consistent with provirus deletion were seen. To confirm recombination, the DNA was digested with HindIII, which cuts upstream of puromycin (FIG. 10A). As expected, when hybridized to the SV40 or puromycin probe, only bands of variable size were seen. In addition, the hybridization pattern was unique, reflecting fragments extending from the Hind III site of the 3 'LTR to sites in the 5' and 3 'flanking cellular DNA, respectively (Figure 10B, data not shown). ). When analyzed by PCR, only 3 out of 12 clones produced weak Cre-specific amplification products (FIG. 10C). 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[Procedure for Amendment] Article 184-8, Paragraph 1 of the Patent Act [Date of Submission] March 18, 1997 [Contents of Amendment] [Fig. 1] FIG. 2 FIG. 3 FIG. 4 FIG. 5 FIG. 6 FIG. 7 FIG. 8 FIG. 9 FIG. 9 FIG. 9 FIG. 10 FIG. 10 FIG. 10

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, M C, NL, PT, SE), OA (BF, BJ, CF, CG , CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (KE, LS, MW, SD, SZ, U G), UA (AZ, BY, KG, KZ, MD, RU, TJ , TM), AL, AM, AT, AU, AZ, BB, BG , BR, BY, CA, CH, CN, CZ, DE, DK, EE, ES, FI, GB, GE, HU, IS, JP, K E, KG, KP, KR, KZ, LK, LR, LS, LT , LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, S G, SI, SK, TJ, TM, TR, TT, UA, UG , US, UZ, VN (71) Applicant Gretz, Manuel             Germany Day 60596 Frank             Furt am Main, Paul-Ehrli             Luch-Strasse 42 (72) Inventor Huon Melfner, Harald             Germany Day 60590 Frank             Furt am Main, Theodor-Ste             Run Kai 7, Unifertita Tsukuri             Nikum Frankfurt Apt. Hamato             Roggy (72) Inventors Rus, Andreas, Peter             Federal Republic of Germany-60590 Frank             Furt am Main, Theodor-Ste             Run Kai 7, Unifertita Tsukuri             Nikum Frankfurt Apt. Hamato             Roggy (72) Inventors Gretz, Manuel             Germany Day 60596 Frank             Furt am Main, Paul-Ehrli             Luch-Strasse 42

Claims (1)

[Claims]   1. At least one coding sequence for a site-specific recombinase; Genetic comprising at least one target sequence specifically recognized by a combinase Vector system useful for child therapy.   2. The recombinase is Cre and the target sequence is loxP. A vector system according to claim 1.   3. The recombinase is Flp and the target sequence is frt. The vector system according to the paragraph.   4. A vector system according to claims 1 to 3, comprising an additional transcription unit.   5. Claims 1 to 4 which are retroviral DNA or RNA. The vector system according to any one of the preceding claims.   6. The method according to claim 5, wherein the target sequence is contained in the U3 and / or U5 region. The vector system shown.   7. Claim 5: The additional transcription unit is in the U3 and / or U5 region. Item 7. The vector system according to item 6 or 6.   8. Claims further comprising a viral promoter and / or enhancer. A vector system according to any of paragraphs 1 to 7.   9. At least one target sequence according to claim 1, claim 4, claim 4. Containing at least one foreign transcription unit according to claim 1 and comprising in its genome A mammal, wherein the vector system is essentially free of other vector sequences according to paragraph 1. Animal cells.   10. 10. The mammalian cell according to claim 9, wherein the target sequence is loxP.   11. 10. The mammalian cell according to claim 9, wherein the target sequence is frt.   12. A method for introducing a cDNA sequence into a mammalian genome, comprising: -The retroviral vectors according to claims 1 to 8 basically by standard methods. The process of making a reactor, Transfecting the producer cells, -Selecting a virus-producing clone; -Infecting mammalian cells, -Expressing a site-specific recombinase, -Includes simultaneous deletion of recombinase expression cassette and most proviral sequences A recombination step between two or more specific target sequences, The above method, comprising:   13. A method according to claim 12, comprising the following steps: -The retroviral vectors according to claims 1 to 8 basically by standard methods. The process of making a reactor, -Infecting mammalian cells, -Expressing a recombinase, -Includes simultaneous deletion of recombinase expression cassette and most proviral sequences Recombination step between two or more specific target sequences.   14． A vector system according to any one of claims 1 to 8, Incorporating nucleic acid into mammalian cell genome, including the step of introducing the nucleic acid into a target cell Law.   15. Claims (1) for incorporating a nucleic acid into the genome of a mammalian cell. Use of the vector according to any one of items 1 to 8.   16. The vector according to any one of claims 1 to 8 as a medicine. ー system