DE19503952A1 - Retroviral vector hybrids and their use for gene transfer - Google Patents

Abstract

The disclosure relates to replication defective retroviral vector hybrids characterized by the fact that they contain: ( a) in the leader region as U5 region and/or tRNA primer binding site, the U5 region and/or tRNA primer binding site of MESV; and (b) as U3 and R regions in 3'-LTR, the U3 and R regions from a Friend murine leukaemia virus (F-MuLV). The proposed hybrids are particularly suitable for effective gene transfer in haematopoietic stem cells.

Description

The invention relates to retroviral vector hybrids and their use for gene transfer, especially for gene transfer in hematopoietic stem cells (ES).

Replication-defective retroviral vectors are currently standard in gene transfer and Gene therapy applications on cells of the human hematopoietic system (A.D. Miller (1992) (1), R.C. Mulligan (1993) (2), R. Vile and S.J. Russell (1994) (3)). Your essential The advantages are:

• - The infection leads to a stable integration of the vector and through it transferred DNA sequences into the host genome of mitotically active cells
• - The number of integrations into the host genome is beyond the infection conditions controllable
• - the safety-related aspects of retroviral gene transfer have been well researched; So far, complications have not occurred in numerous human applications.

The most frequently mentioned advantage of retroviral vectors, the high gene transfer efficiency, applies use on the hematopoietic system is limited. With conventional, on the Moloney murine sarcoma virus (MoMuSV) based vectors are preferred only infecting late, maturing hematopoietic progenitor cells; 30-95% of those using these vectors Transduced progenitor cells show no or due to primary silencing mechanisms insufficient expression of the transferred genes (4); after a long stay in vivo also becomes retroviral in a high percentage of the initially expressing cells controlled gene expression up to inability to function through secondary silencing weakened (Palmer et al. (1991) (5), Brenner et al. (1993) (6)).

It has been shown that even in embryonic cells, such as embryonic carcinoma cells (myeloid (non-lymphatic) descendants of hematopoietic stem cells), the Viruses are integrated, but the expression of the integrated provirus is blocked.

From Stocking et al. (1993) (21) mutations and derivatives of MoMuSV are known with which can improve retroviral gene expression in such cells. Such  Hybrids are caused by point mutations in the LTRs, especially in the U3 region receive. For example, a point mutation at -345 of MoMuLV can bind of the ECF-1 repressor can be reduced. A point mutation, which is also advantageous creates a binding site for SP-1 (e.g. point mutation at -166 (5. McKnight and R. Tÿan (1986) (16)). It is also advantageous to make mutations outside the LTR areas to attach. It has been shown that an 18 bp region directly downstream of the 5′- LTR connects, represents a negative regulatory element (NRE, silencer binding element). Point mutations in this element make it possible to increase retroviral transcription improve (murine embryonic stem cell virus, MESV; M. Grez et al (1990) (22)).

The object of the invention is to provide retroviral vector hybrids with which the retroviral gene expression especially in hematopoietic stem cells and their myeloid (non-lymphatic) descendants can be further improved.

The invention relates to retroviral vector hybrids, which are characterized in that that she

• a) in the leader region as the US region and tRNA primer binding site the US region and tRNA primer binding site from MESV and
• b) as U3 and R regions in 3'-LTR, the U3 and R regions from a friend murine leukemia virus (F-MuLV)

contain.

The vector hybrids according to the invention preferably contain the leader region Leader region of MESV.

The vector hybrids according to the invention likewise preferably contain the LTR as the 3′-LTR a friend murine leukemia virus (F-MuLV) and thus, except for the U3 and R regions MoMuLV, also the US region.

The vector hybrids can be replication-defective as well as replication-competent.

A replication-defective vector is a vector that is not retroviral Contains gene functions (gag, env, pol) and therefore do not form virus particles independently can. However, the vector usually contains a packaging function (psi). For education infectious retroviral particles become a packaging cell line that Contains functions gag env and pol stable (as an episome or integrated into the genome) with the  replication-defective DNA vector according to the invention transfected. RNA- Transcripts formed that are packaged in virus particles due to the gag and env functions will. These virus particles are replication deficient because the RNA Genome does not carry retroviral gene functions.

Replication-capable, retroviral vector hybrids additionally contain the gene regions gag; pole and env. These gene regions can consist of any retroviruses. Only the env- Region must be selected according to the cell to be infected. Such procedures are known to the expert. In this way, replication-capable ecotropic, xenotropic, amphotropic or polytropic retroviruses can be produced.

More on this as well as on the production of vectors, virus particles and helper cells is described in M. Kriegler, Gene Transfer and Expression, A Laboratory Manual, W.H. Freeman & Co., New York (1990), 47-55 and 161-164) (31). This publication is Subject of the disclosure of the present application.

Among silencing proteins in the sense of the invention are cellular (e.g. from the mutated cell derived) nuclear proteins, for example from hematopoietic stem cells understand which preferably to the first 450 bp immediately following the 5'-LTR, preferably bind the first 18 bp, of MoMuLV and MoMuSV and by interaction with retroviral transcription with cis-regulatory sequences of the provirus.

Leader region of MESV is to be understood as the sum of sequences from the short direct terinal repeat (R), the US region and the sequence immediately following the 5′-LTR of MESV, which includes both the tRNA primer binding site and the packaging Region (ψ) contains. The leader sequence can consist at most of the complete sequence which is contained in the virus from R to the start of translation. However, a shortened leader region is preferably used which, in addition to R and US, preferably consists of approximately the first 450 bases downstream from the end of the 5'-LTR region. It is also preferred that only or essentially the US region and the tRNA primer binding site from MESV and the other parts of the leader sequence originate from MoMuLV or MoMuSV. A suitable MESV range is shown in the illustrations ( Fig. 1-5) from KpnI (approx. 550) to BalI (approx. 729).

The leader region of MESV corresponds to the leader region of MoMuLV or MoMuSV (Sequence of U3, US and R of MoMuLV and PCMV, see (22) and (23)), in the  Point mutations are inserted with which the binding of silencer proteins is reduced or is prevented. The leader region of MESV contains MoMuLV and PCMV in the 18 bp region, which directly adjoins the 5′-LTR, 5 point mutations (C. Stocking et al. (1993) (21)). The inhibitory binding ability of Core proteins from the infected lines were drastically reduced and the expression of an im Vector hybrid contained gene accordingly improved (L Petersen et al., Mol. Cell. Biol. 11 (1991) 1213-1221 (8)). Instead of these point mutations there are also other (at least one) Point mutations in the leader region (preferably within the first 450 bases downstream from the end of the 5′-LTR region, and particularly preferably in the 18 bp region) suitable, the inhibitory binding of silencer proteins z. B. to the leader region reduce or prevent. This is in the spirit of the invention under a leader region of MESV in addition to the sequence mentioned above and described in SEQ ID NO: 1 also a To understand the sequence which essentially corresponds to this section of SEQ ID NO: 1, contains a tRNA binding site and a psi function and so through point mutations is modified that the inhibitory binding of silencer proteins from the cells which are used for Transfection are provided, is reduced or prevented.

To check whether a point mutation is suitable, the binding of silencer proteins to prevent the leader region, a plasmid is usually produced which contains an LTR, contains the leader region to be checked and an indicator gene, preferably the CAT gene. The lines to be checked are transfected with this plasmid and the CAT activity determined after preferably 48 hours (transient transfection). Is the CAT activity the point mutation is clearly present, the binding of the silencer protein in to prevent these cells (P. Artelt et al. (1991) (32)).

Transient transfection experiments show that the leader sequence of MoMuSV cis-regulatory sequences are localized, which have an inhibitory influence on have gene expression especially in early, multipotent myeloid cells (primary Silencing). Exchange of this area for leader sequences of the murine embryonic Stem cell virus [MESV (22)] increases the inhibitory influence in myeloid stem cells completely on. The leader sequence of MESV goes back to the endogenous Mouse Retrovirus dl-S87rev (23) and was introduced in MESV to that on the MoMuSV leader observed silencing in embryonic stem cells (22). Crucial for that Abolition of silencing are mutations of the retroviral primer binding site (PBS) that is located 3 'of the 5'LTR (22).  

Retroviruses of the F-MuLV group are known to the person skilled in the art and are, for example, in D. Linnemeyer et al. (1981) (7), J. Friel et al. (1990) (9), S.P. Clark and T.W. Mak (1982) (10), L. Wolff et al. (1985) (11), R.K. Bestwick et al. (1984) (12), W. Koch et al. (1984) (13) and k Adachi et al. (1984) (14). An overview can be found in W. Ostertag et al. (1987) (15). The 3'-LTRs are preferably from the malignant histiosarcoma virus (MHSV) (9), SFFVp Lilly-Steeves (10), SFFVa (11), Rauscher SFFV (12), F-MuLV c157 (13) and Friend-nunk cell focus forming virus (F-MCFV FrNx (14), F-MCFV pFM548 (13)) used.

According to the invention, 3′-LTR sequences of the genomic vector RNA are suitable, which in the U3 region a high-grade sequence homology to the 3′-LTR areas of the show above friend virus family. The LTR sequence of SFFVp is in SEQ II) NO: 1 (nucleotides 1707-2283).

The advantages according to the invention can be seen in transient transfections of Reporter gene constructs in a variety of representative human and cell lines Mouse. Surprisingly, it has been shown that U3 sequences from mouse retroviruses Friend murine leukemia virus (F-MuLV) family in combination with leader sequences (preferably tRNA binding site) from MESV a particularly efficient gene expression in enable myeloid stem and progenitor cells. The increase in gene expression with vector hybrids according to the invention compared to MoMuSV is more than a power of ten. This applies to both relatively late granulocyte, macrophage and progenitor cells Erythrocytes as well as for early, multipotent precursors and also stem cells of myeloid system too. Especially in the immature, multipotent myeloid cells which MoMuSV-LTR shows a pronounced primary silencing, allow vector hybrids according to the invention an efficient gene expression. Even in the lymphatic System as well as in fibroblasts the activity is high.

An early isolate of the polycythemia-inducing spleen focus is particularly preferred forming virus (SFFVp (7)), whose LTR sequence is shown in SEQ ID NO: 1 (bp 2654-3230) and the malignant histiosarcoma virus (MHSV (9), LTR SEQ ID NO: 4, bp 1707-2324). In the U3 region of these viruses there is a high degree of sequence homology other representatives of the friend virus family such as SFFVp Lilly-Steeves (10), SFFVa (11), Rauscher SFFV (12), F-MuLV c157 (13) and Friend-mink cell focus forming virus [F-MCFV FrNx (14), F-MCFV pFM548 (13)]. This and similar friend-related retroviruses indicate  an expression pattern similar to that due to the sequence homology of the U3 regions SFFVp and MHSV.

Particularly advantageous for the marked increase in the compared to MoMuSV Gene expression in myeloid stem and progenitor cells by the invention Vectors are mutations in the enhancer region of U3 [340 to 140 nucleotides 5 'des Transcription starts located (10)]. The partial duplication of the so-called direct-repeat elements at SFFVp (similar to F-MCFV FrNx) and MHSV. Binding sites for transcription factors that are located in myeloid cells ensure efficient gene expression.

Immediately 5 ′ of the direct repeat carry SFFVp and MHSV as well as Friend MCFV Binding site for the transcription factor Spl (16). Spl is about having multiple interactions surrounding transcription factors that usually led to a significant increase of gene expression. Myeloproliferative sarcoma virus for the MoMuSV derivatives (MPSV (17), (18)) and PCC4 cell-passaged MPSV (PCMV (19)) is the occurrence of one Spl binding site in the U3 region is also necessary for the abolition of silencing of the MoMuSV LTR in embryonic stem cells (20) and probably also in Blood stem cells (21). An analog function could also be the Spl binding site of SFFVp, MHSV and Friend MCF viruses.

Any U3 and R regions can be used in 5′-LTR since after the integration of the virus, the U3 from 3'-LTR in the target cell after the retroviral life cycle is copied to the 5'-LTR position and drives gene expression. U3 and R regions come, for example, from MoMuLV or MoMuSV derivatives, such as MPSV, PCMV and MESV. Also suitable as 5'-LTR are 5'-LTRs from F-MuLV.

The vector hybrids according to the invention show after retroviral transduction of myeloid Stem and progenitor cells have a high tissue-specific expression. Have these vectors hence the potential for gene transfer efficiency in myeloid stem and progenitor cells to increase significantly compared to MoMuSV vectors. The vectors according to the invention have also the potential, to a much lesser extent, of silencing processes in myeloid Cells to be subjected as MoMuSV vectors. The high and possibly also Persistent gene expression of these vectors in myeloid cells can be the basis create for the successful application of numerous gene transfer protocols on the hematopoietic  Human system. The constructs described in the examples are constructed so that the cDNAs transferred from the vector can easily be replaced by other genes.

The tissue specificity of the expression is suitable, that of conventional MoMuSV vectors observed primary silencing in the myeloid system. This leads to a significant increase in functional gene transfer rates in myeloid cells. Because a high functional gene transfer rate in most gene transfer / gene therapy applications on myeloid system is desired, there is a generally usable advantage of constructs according to the invention.

The friend virus-related U3 regions were also found in the mouse myeloid system Persistence of gene expression selected in vivo. Secondary silencing occurs with these Vectors to a much lesser extent than with MoMuSV vectors. The exclusion of inhibitory leader sequences by exchange for MESV sequences be an additional advantage in this regard. This is important for all gene transfer / Gene therapy applications in which long-lasting (possibly lifelong) expression the retrovirally transmitted sequences are desired in the myeloid system (Gene labeling studies, correction of metabolic defects).

For this, the vector hybrids according to the invention can have at least one for the virus heterologous gene that can be expressed in eukaryotic cells. Such genes are for example the multiple drug resistance gene (MDR gene), an antibiotic resistance gene such as the neo® gene, the LNGFR gene, the cerebrosidase gene or the herpes simplex TK gene. In In a preferred embodiment, several, preferably two or three, can also be used heterologous genes can be contained in the vector hybrid. To express these separate genes To make it possible, a promoter, a splicing, is expediently placed in front of the second or third gene site (preferably from SF7Vp) or an internal ribosomal entry site (IRES, preferably of polio viruses (I.R. Ghattas et al. (1991) (26))).

The generation of high virus titers in fibroblastoid packaging cell lines is another important requirement for retroviral vectors for gene transfer into myeloid cells should be used. The activity of friend-related U3 regions in retrovirus fibroblastic packaging cell lines are sufficient for those listed below Example constructs in any case, the required titer from 105 to 106 to generate vector-transmitting retroviral particles / ml of cell culture supernatant.  

The invention accordingly also relates to a method for producing a retroviral transduced eukaryotic cell containing an active exogenous gene which characterized in that the eukaryotic cell is transduced with a replication-defective, retroviral vector virus, which in its genome as

• a) Leader region the MESV leader region,
• b) as U3 and R regions in 3'-LTR, the U3 and R regions from a friend murine leukemia virus (F-MuLV) and
• c) contains said exogenous gene.

Preferably, the LTR from F-MuLV is used as the 3'-LTR, which is then the one mentioned Contains U3 and R regions.

The invention further relates to retrovirally transduced, eukaryotic cells, obtainable by transducing the eukaryotic cell with a replication-defective, retroviral vector virus, which in its genome as

• a) Leader region the leader region of MESV and
• b) as U3 and R regions in 3'-LTR, the U3 and R regions from a friend murine leukemia virus (F-MuLV) contains.

Optionally, this vector contains one or more (up to three) exogenous genes that are in the eukaryotic cell are expressible. Preferably used as eukaryotic cells Mammalian cells, preferably hematopoietic cells, especially hematopoietic cells Stem cells used.

Preferably, the LTR from F-MuLV is used as the 3'-LTR, which is then the one mentioned Contains U3 and R regions.

An active exogenous gene is to be understood as a gene that enters the cell from the outside is introduced and is expressed in this cell after integration into the genome (is active). The exogenous gene can be a gene not contained in the cell genome (e.g. a Antibiotic resistance gene such as neoR, LNGFR receptor (D. Johnson et al. (1986) (33)), the Cerebrosidase gene or the herpes simplex TK gene) or one contained in the genome, but there is no or only insufficiently expressed gene there, such as multiple drug resistance (MDR) gene.

Another object of the invention is a replication-defective, infectious virus particle, which contains a retroviral RNA as the genome, the genome comprising a leader region  MESV, which contains a packaging function and a t-RNA binding site, contains an in a eukaryotic cell that is expressible and heterologous for the virus and at the 3'-end U3 and R from a Friend murine leukemia virus (F-MuLV), but no active gag, env and contains pol sequences.

Another object of the invention is a method for producing a replication-defective, infectious virus particles by transfection of a eukaryotic Helper cell, which has the helper functions gag, env and pol, with a vector hybrid, which as leader region is the leader region of MESV and as 3′-LTR the LTR from one Friend murine leukemia virus contains, production of the DNA of the vector hybrid corresponding RNA as a virus genome in the cell (for example through cellular Polymerases), packaging of the RNA mentioned in the cell, replication-deficient, empty virus envelopes and isolation of the infectious virus particles, which the contain called virus genome.

Another object of the invention is the use of an inventive Replication-defective, retroviral vector hybrids, which is the leader region from MESV and as U3 and R regions in 3′LTR, the U3 and R regions from a friend contains murine leukemia virus (F-MuLV), for the manufacture of a medicinal product for ex vivo or in vivo gene therapy.  

application areas I. All sornatic gene transfer / gene therapy procedures in which myeloid stem and Precursor cells are target population for retroviral vectors

The individual protocols are each different cDNAs in the vectors integrate. examples are

• - Gene labeling studies (1): selectable marker genes such as neoR or shortened nerve growth factor receptor are transmitted to the fate of the labeled cell population in the Organism under the conditions of the disease examined in each case follow. The neoR vectors pSF1N and pMH1N, the neoR under the control of SFFVp U3 or wear MHSV U3 (MESV leader) show in comparison to neoR-transmitting Moloney vectors a significant increase in the functional gene transfer rate in the Model system of the mouse stem cell line FDCPmix. They are for the neoR transfer to humane myeloid stem cells.
• - Protection of the bone marrow against the side effects of high-dose chemotherapy (28): Chemotherapy resistance-mediating genes such as MDR1 or alkyl transferases transfer. High transfer rate and expression via the optimized friend virus-related Vectors prevent myelosuppressive side effects and possibly also secondary tumor induction of chemotherapy.
• - Correction of metabolic diseases: With monogenic hereditary diseases are over retroviral gene transfer to myeloid stem cells intact copies of the defective genes brought in. Applications are e.g. B. conceivable for suffering, whose genetic defect effects in the myeloid system (storage diseases such as Hurler syndrome; M. Gaucher (29)):

II. Cloning of replication-competent friend-related retroviruses

The viruses of the invention are due to their early myeloid strain and progenitor cells expanded host spectrum particularly well for insertion mutagenesis in suitable for these cells. Insertion mutagenesis can be used to clone genes, the one important function in the physiological growth regulation of the myeloid system to have.  

The principle of using retroviruses as insertion mutagen has been used several times (Review: Kung et al. (1991) (34)). Retroviruses integrate DNA sequences non-specific into the genome and can activate or deactivate genes. Replicable Retroviruses infect tissues with a very high efficiency and can therefore Increase the likelihood that mutations will be created by integration. Such Mutations can in turn lead to degeneration of the mutated cells, i.e. H. Tumors arise.

In in vivo experiments with mice (usually on newborn mice since they do not yet have any have an efficient immune system and are therefore very sensitive to retroviruses) to identify regulatory genes, those are infected with retroviruses, and after one Latency period of approx. 3 months can specific tumors (depending on the virus type) discovered and examined. Analyzes of the clonal, retroviral integration sites reveal genes, the activation or inactivation of which changed or degenerated into cells Phenotype. This makes it possible to apply a large number of different types of genes discover how genes for ligands, receptors, signal transducers and Transcription factors. Significantly, there is a high correlation between the found tumor type, which is produced by certain viruses, and the activatedfractivated gene (e.g. in erythroleukemia induced by Friend-MuLV 80% of the tumors show integration events in the Fli-1 locus and transcriptional Activation of the transcription factor PU-1 gene: Interaglerende Oncogene could can be identified by transgenic mice with ecotropic expression of an oncogene (e.g. myc) and the tumorigenesis was observed. After a significantly reduced latency tumors were caused by secondary mutations ("Seeond Hit", eg in the pim-1 gene)).

Nevertheless, only a selection of possible ones could be made with previous retrovirus constructs Genes are identified. The range of genes that go through with this approach Activation / inactivation and the associated phenotypic change in the cell, is heavily dependent on the specificity of the retroviruses used for certain Cell types. Retroviruses used so far show very little infectiousness in hematopoietic and embryonic stem cells (see review: Stocking et al. (1993) (21)). There are several reasons for this; primarily on transcription control elements in the retroviral LTRs (Stocking et al. (1985) (18)), but also secondarily for which Virus replication necessary, protein interactions between cellular and viral proteins.  

Replication-competent retroviruses create new ones through infection Retrovirus producer cells, leading to complete infection of the whole Cell population and is much more likely to develop Mutations. Similar efficiency can only be achieved through a more complex and time-consuming process Reach cocultivation of the retrovirus packaging line with the target cell line.

Friend-related replication-competent viruses can also be used to generate forced passage mutants are used, which further improve the infectability of blood stem cells. Mutations in retroviral structural proteins of these viruses can be used in the production of optimized packaging cell lines for the Gene transfer to myeloid cells. Mutations in cis-regulatory sequences of these viruses can be used for further optimization of retroviral vectors for the Blood stem cell gene transfer.

The invention further relates to replication-capable, ecotropic, xenotropic, amphotropic or polytropic retroviruses, with the regulatory elements (leader region) from MESV and possibly MoMuLV or MoMuSV, combined with U3- and R- Regions in 3′-LTR, the U3 and R reglones from a Friend murine leukemia virus (F- MuLV), which are able to produce murine (in vivo and in vitro) or human (in vitro) Progenitor cells more efficiently to trigger inner and mutations. Preferably included the retroviruses as 3′-LTR the LTR from F-MuLV.

The combination of both components leads to retroviral insertion mutagenesis constructs a new quality, with the help of new, with conventional, replication-capable retroviruses genes not found, can be identified. They are with a very high titer can be generated and result in an almost 100% infection rate, since each infected cell is used for Producer cell for new viruses. The neomycin resistance gene, which is found in retroviral vectors for selection are used, deleted. This leads to a higher transcription of the LTR promoters, since neoR functions as a silencer (Artelt et al. (1991) (32)), and consequently to an improved insertion rate.

The retroviruses according to the invention allow murine and human hematopoietic and embryonic stem cells infected in vitro with high efficiency and by insertion into the Genome mutants (activation or inactivation of genes) are generated. It is therefore also expected for in vivo approaches that the viruses according to the invention are early  hematopoietic and embryonic lines infect and genes with greater efficiency Activate or deactivate integration into the genome.

Gene activation can happen in several ways. So can an integration upstream of a gene that under the transcriptional control of the 3'-LTR promoter activate. It can also inactivate the binding sites of negative control elements what can lead to gene activation. However, there are also several examples (Kung et al. (1991) (34)), who prove that the 5'-LTR promoter can be read directly or but because of its enhancer effect also many bases upstream, but also within one Introns, after insertion, can activate a gene.

Integration within a gene or in the associated promoter / enhancer region can also lead to inactivation of the corresponding gene.

If genes that are important for the regulation of the cell are affected, the cells can degenerate. H. it can cause tumors. With the help of the viruses according to the invention, others can therefore Genes, e.g. B. genes involved in cell regulation of hematopoietic or embryonic Stem cells are involved than with conventional viruses found in those Can not replicate or only very poorly replicate cell types.

The replication-competent viruses according to the invention can be used in vitro, for example, as follows:
Factor-dependent, promyelocytic cell lines were made factor-independent by retroviral insertion mutagenesis, ie the growth factor gene was activated by retroviral integration. The retrovirus infection was carried out by coculturing the target cell line with packaging lines producing MPSV vector. This in vitro approach can be made more efficient with the help of the constructs according to the invention. Furthermore, murine or human, embryonic and hematopoietic stem cells are accessible with the viruses according to the invention.

Another application is in experiments that aim to do better Vector virus packaging lines e.g. B. for gene therapy, the viruses with a can produce higher infection efficiency in progenitor cells. Exist in those cells several blocks that interfere with the retroviral infection / replication. The usage of the constructs according to the invention has MoMuLV or  AM4070 the advantage that the transcriptional blocks are already removed. Improvements In the efficacy of infection, corresponding mutations can therefore be found in the viral Return proteins.

Another possible application of the constructs according to the invention is in Generation of "knock out" mice by inactivating one or more genes in embryonic stem cells.

The following examples, drawings and sequence listings further illustrate the invention.

The sequence listings show:
SEQ ID NO: 1 sequence of pSF1
SEQ ID NO: 2 sequence of pSF2
SEQ ID NO: 3 sequence of pSF3
SEQ ID NO: 4 sequence of pMM1
SEQ ID NO: 5 sequence of pSF-MDR (only the proviral part of the sequences is shown)

Figure 1A shows the plasmid map of pSF-MDR. (indicative information about the restriction sites)

FIG. 1B shows the plasmid map of pSF-MDR (accurate indication of restriction sites)

Figure 2A shows the restriction map of pSF1.

Figure 2B shows the restriction map of pSF1N. This vector corresponds to pSF1 into which the neoR gene has been inserted into the multiple cloning site (MCS).

Figure 3A shows the restriction map of pSF2.

Fig. 3B shows the restriction map of pSF2N which pSF2 corresponds to integration of the neo R gene.

Figure 4A shows the restriction map of pSF3.

FIG. 4B shows the restriction map of pSF3N which pSF3 corresponds to integration of the neo R gene.

Figure 5A shows the restriction map of pMH1.

Figure 5B shows the restriction map of pMH1N, which corresponds to pMH1 after integration of the neoR gene.

Fig. 6 shows the formation scheme of the vector pSF-MDR1.

Fig. 7 shows the formation scheme of the vectors pSF2N, pSF2N, pSF3N and pMH1N.

example 1 a) Construction description Table 1 shows the cloning strategies of the constructs according to the invention I. pSF-MDR ( Fig. 1, 6, Table 1, SEQ ID NO: 5)

This vector was cloned on the basis of the MESV vector R224 (base pUC 19), in which parts of the env region and the complete U3 region of the 3′LTR were replaced by SFFVp sequences. In this way, the vector pSFs + 3 (see FIG. 6) was obtained. In a second step, the cDNA coding for MDR1 (from pMDR2000XS (25)) was inserted into the multiple cloning site (Not1, Xba1, BamH1, Hind3).

II. PSF1N, pSF2N, pSF3N, pMH1N ( Fig. 2-5, 7, Table 1)

The backbone of these plasmids is based on the MESV vector R224, in which there was a large deletion between the Xbal interface of the 5'LTR and the Kpnl interface in the 3'LTR. The complementing sequences of the three-fragment ligations were introduced; the base vectors pSF1, pSF2, pSF3 and pMH1 resulted (development scheme and cloning strategy, FIG. 7, Table 1, sequences described in SEQ ID NO: 1-4). The fragments used for the three-tag ligation are obtained from auxiliary constructs (see development diagram, FIG. 6), in which modifications can easily be made to further optimize the vectors. It is conceivable to exchange the SFFVp or MHSV U3 regions for analogue sequences from other friend-related retroviruses or to modify the leader mentioned above to optimize the titer. After insertion of the neoR cDNA (from R229), the sample constructs pSF1N, pSF2N, pSF3N, pMH1N were created. The sample constructs carry polylinkers with unique restriction sites that can be used to exchange the genes to be transferred. Regulatory sequences for a possibly necessary second gene expression such as the splice acceptor of SFFVp or the so-called internal ribosome entry point IRES (IR Ghattas et al. (1991) (26)) can also be introduced at these interfaces.

The friend-related regions cloned as U3 and R in the sample constructs (in 3′LTR) are also copied to the 5′LTR position after the end of a retroviral life cycle and then drive gene expression in the target cells. The sample constructs contain all for retroviral gene transfer and retroviral gene expression necessary cis-regulatory Elements. The properties of SFFVp (or MHSV), MESV and MoMuSV combined. The are integrated as safety-relevant modifications Point mutation of the start codon for the retroviral gag protein to a stop codon (A.D. Miller and G.J. Rosman (1990) (27)) and - for pSF1N, pSF2N, pSF3N, pMH1N - the Deletion of unnecessary env-squences (27).

Table 1

Cloning strategies of the constructs and auxiliary constructs according to the invention

Example 2 Functional gene transfer rate increased compared to conventional Moloney Vectors

The multiple drug resistance 1 (MDR1) gene codes for an efflux pump with a membrane (P-glycoprotein), which mediates resistance to a number of clinically important cytostatics (I. Pastan et al. (1988) (25)). The level of resistance is closely related to the level of expression of P- Glycoprotein linked. If myeloid stem cells are successful with retroviral MDR1 Making cytostatic resistant transfer could make an important contribution to lowering the Side effect rate of tumor chemotherapy can be performed.

In the experiment, K562 cells (ATCC C11243) and TF1 cells (human myeloid Progenitor cell lines) infected with retroviral vectors, the MDR1 under the control of the SFFV Express LTRs (Virus SF-MDR emerged from construct pSF-MDR with MESV- Leader).

The TF1 cell line was as described in T. Kitamura et al. (1989) (30) described by one Receive patients with erythroleukemia. The cells are supplemented in RPMJ medium ( with 20% fetal calf serum, 1 mM Na pyruvate, 4 mM glutamine and 20 U / ml recombinant GM-CSF) kept in culture.

In an identical approach, K562 cells and TF1 cells were infected with a Moloney-MDR1 vector (Virus V-MDR originated from the construct pVMDR), which in a clinical gene transfer protocol headed by Prof. A. Deisseroth at the MD Anderson Cancer Center, Houston, Texas. As can be seen from Table 2, SF-MDR in K562 leads to an approximately 20-fold increase in the gene transfer rate VMDR, measured by the number of cytostatic applications (approx. 10 times LDSO for K562) growing colonies.

The increase in the functional gene transfer rate in myeloid cells is an expression of average increase in gene expression rate in the transduced cells. This is from Importance in gene transfer applications, their success also depends on the level of gene expression depends (see application example: bone marrow protection in high-dose chemotherapy). In the experiment described above (infection of human myeloid cells with SFMDR or V-MDR) the cells are plated under very high cytostatic doses (approx. 20 times  LD50 in TF1), only SF-MDR infected cells are able to form colonies (Table 2).

Table 2

Colony count under colchicine selection after infection of human hematopoietic cells K562 and TF1 by MDR1-tansducing retroviral vectors

The SFFV _p - / MESV vector significantly increases the functional gene transfer rate compared to the conventional MoMuLV vector

The cells were filled with retroviral supernatant from amphotropic packaging cell lines infected. The titer on fibroblasts was for both vectors at the time of the experiment approx. 2 × 104 / ml. The ratio of vector / target cell was <1. 24 hours after infection Infection, 5 × 10⁴ cells were plated in colchicine-containing soft agar medium. The Evaluation was carried out on day 8 of the agar solution. The cloning efficiency without selection was approximately 15% for both cell lines. The experiments were carried out in duplicates.

For a vector whose U3 region comes from MoMuLV and whose leader from MESV, results similar to those obtained with the V-MDR vector are obtained.

In the MDR1 system, constructs with an MESV leader have compared to conventional ones MoMuSV vectors somewhat reduced titers. This could be due to packaging mutations from MESV. Hybrid between the MESV and MoMuSV have the potential to improve vectors with higher titer due to Packaging function in retroviral packaging cell lines with simultaneous exclusion cis to generate inhibitory sequences. This was when the sample constructs were cloned pSF2N and pSF3N taken into account.  

Example 3 Construction of replication-capable insertion mutagenesis vectors based on the Friend-JMESV vector constructs according to the invention

The example construct of a replication-capable, amphotropic Friend / MESV vector for insertion mutagenesis was produced by cloning the gag-pol-env genes necessary for retroviral replication in the Friend / MESV vector pSF1 and pSF3 (see FIGS . 2A and 4A).

For this purpose, the entire 7.2 kb gag-pol-env gene cassette made of pAM (Miller et al. (1985) (35)) using the "Expand long template PCR system" (Boehringer Mannheim, see also: W.M. Barns (1994) (36)) amplified and incorporated into pSF1 and pSF3.

pAM represents a fusion construct from the ecotropic MoMuLV vector pMLV-K (gag-pol to SalI interface) and from the amphotropic virus 4070A vector p4070A (pol from SalI interface and envamphotropic). The upper PCR primer was 60 bp upstream of the gag start codon (directly adjacent to the PstI interface), the lower PCR primer placed directly at the end of the env ^amphotropic gene. The 7.2 kb gag-pol-env ^amphotropic PCR amplificate was cut uncut, ie with blunt ends, into those cut with PstI and Hind and treated with Klenow enzyme to remove the overhangs (Sambrook et al. (1989) (37)) ) Vector pSF1 and pSF3 built in. The resulting vectors were given the names pSF1-AM, pSF3-AM and have the following basic structure: 5'-LTR ^MESV - PBS (-) ^MESV -gag-pol-env ^amphotropic -3'-LTR ^SFFV (pSF1-AM) and 5 ′ -LTR ^MESV - PBS (-) ^MUSV -gag-polenv ^anphotropic -3′-LTR ^SFFV (pSF3-AM). After transfection into human or murine cells, they are able to produce replication-competent retroviruses (RCR), which can be used for insertion mutagenesis in hematopoietic stem and progenitor cells.

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SEQUENCE PROTOCOL

Claims (28)

1. Retroviral vector hybrid, characterized in that it

• a) in the leader region as the US region and tRNA primer binding site the US region and tRNA primer binding site of MESV or MoMuSV and
• b) as U3 and R regions in 3'-LTR, which contains U3 and R regions from a Friend murine leukemia virus (F-MuLV).

2. Retroviral vector hybrid according to claim 1, characterized in that it as a 3'-LTR contains the LTR from F-MuLV. 3. vector hybrid according to claims 1 or 2, characterized in that it as Leader region contains the leader region of MESV. 4. vector hybrid according to claims 1 to 3, characterized in that the 3'-LTR from the malignant histiosarcoma virus (MHSV), SFFVp Lilly-Steeves, SFFVa, Rauscher SFFV, F-MuLV c157 or Friend-mink cell focus forming virus (F-MCFV FrNx, F- MCFV pFMs48) is used. 5. vector hybrid according to claims 1 to 4, characterized in that as U3 and R Regions the U3 and R regions from MPSV, MESV or PCMV are used. 6. Vector hybrid according to claims 1 to 4, characterized in that the 5'-LTR LTR from a Friend murine leukemia virus (F-MuLV) is used. 7. vector hybrid according to claims 1 to 6, characterized in that as 5'-LTR bp 142-657 from SEQ II) NO: 1 and / or as 3'-LTR bp 1707-2283 from SEQ II) NO: 1 is used. 8. vector hybrid according to claims 1 to 7, characterized in that it is at least contains a gene which is heterologous to the virus and which can be expressed in eukaryotic cells. 9. vector hybrid according to claim 8, characterized in that it is a heterologous gene multiple drug resistance gene (MDR gene), an antibiotic resistance gene like the neo® Gene, the LNGFR gene, the cerebrosidase gene or the herpes simplex TK gene holds.   10. Vector hybrids pSF1, pSF2, pSF3, pHM1. 11. Replication defective vector hybrid according to claims 1 to 10. 12. Replication-competent vector hybrid according to claims 1 to 7. 13. A method for producing a retrovirally transduced, eukryotic cell, which contains an active exogenous gene, characterized in that the eukaryotic cell is transduced with a retroviral vector virus, which is in its genome

• a) in the leader region as the US region and tRNA primer binding site, the US region and tRNA primer binding site of MESV or MoMuSV,
• b) as U3 and R regions in 3'-LTR, the U3 and R regions from a Friend murine leukemia virus (F-MuLV) and
• c) contains said exogenous gene.

14. A method for producing according to claim 12, characterized in that the retroviral vector virus as 3'-LTR which contains LTR from F-MuLV. 15. The method according to claims 12 or 14, characterized in that as Leader region the leader region is used by MESV. 16. The method according to claims 12 to 15, characterized in that a replication-defective, retroviral vector virus is used. 17. Retrovirally transduced eukaryotic cell, obtainable by transducing the eukaryotic cell with a retroviral vector virus found in its genome

• a) in the leader region as the US region and tRNA primer binding site the US region and tRNA primer binding site of MESV and
• b) as U3 and R regions in 3'-LTR, which contains U3 and R regions from a Friend murine leukemia virus (F-MuLV).

18. Cells according to claim 17, characterized in that the retroviral vector virus in his genome as 3'-LTR contains the LTR from F-MuLV.   19. Cell according to claims 17 or 18, characterized in that as the leader region the leader region is used by MESV. 20. Cell according to claims 17 to 19, characterized in that a replication-defective, retroviral vector virus is used. 21. The method according to claims 13 to 16, characterized in that as eukaryonti hematopoietic stem cells are used. 22. Replication-defective, infectious virus particle, which as a genome is a retroviral RNA contains, the genome as the US region and tRNA primer binding site the US region and tRNA primer binding site from MESV or MoMuSV, a packaging function in a eukaryotic cell expressible and for the virus heterologous gene and am 3′-end U3 and R from a Friend murine leukemia virus (F-MuLV), but none contains active gag, env and pol sequences. 23. A method for producing a replication-defective, infectious virus particle Transfection of a eukaryotic helper cell, which gag the helper functions, env and pol owns, with a vector hybrid which is in the leader region as a US region and tRNA primer binding site the US region and tRNA primer binding site from MESV and as U3 and R regions in 3′-LTR, the U3 and R regions from a friend murine leukemia virus (F-MuLV) contains, production of the DNA of the vector hybrid corresponding RNA as virus genome in the cell, packaging of the virus genome in replication deficient, empty virus envelopes and isolation of the infectious virus particles that contain the named virus genome. 24. The method according to claim 23, characterized in that the LTR as 3'-LTR F-MuLV is used. 25. The method according to claims 23 or 24, characterized in that as Leader region the leader region is used by MESV. 26. Use of a replication-defective, retroviral vector hybrid, which in the Leader region as the US region and tRNA primer binding site the US region and tRNA primer binding site from MESV and as U3 and R regions in 3′-LTR, the U3 and  Contains R regions from a Friend murine leukemia virus (F-MuLV) for the production a drug for ex vivo or in vivo gene therapy. 27. Use of a vector hybrid according to claim 26, characterized in that it as 3'-LTR the LTR from F-MuLV is used. 28. Use according to claims 26 or 27, characterized in that as Leader region the leader region is used by MESV.