DE19808438A1 - Retroviral vectors, processes for their preparation and their use for gene transfer into CD4-positive cells - Google Patents

Abstract

The invention relates to the production and use of retroviral vectors for cell-type specific gene transfer, specially to a production method of retroviral vectors containing capsid particles of murine leukemia virus (MLV) and envelope proteins of human immunodeficiency viruses (HIV) or simian immunodeficiency viruses (SIV). Said vectors can be used for gene transfer in selected cell types, specially in CD4-positive mammal cells.

Description

The invention relates to retroviral vectors (cell targeting vectors), methods for their production and their use for gene transfer into CD4 positive cells.

The term "retroviral vectors" or "retroviral transfer vectors" denotes infectious but retroviruses unable to reproduce, the genes in the form of retroviral expression constructs (also called expression vectors) into cells. The gene transfer leads to Integration of the expression construct into the genome of the cell. The retroviral gene transfer is advantageous because (i) a copy of the desired gene is usually converted into cells, (ii) the gene is generally transferred without mutations or rearrangements and (iii) a stable chromosomal incorporation takes place.

It is known to use retroviral vectors based on the amphotropic murine leukemia virus (MLV) to use to target certain genes in mammalian cells, particularly in human cells convict. These vectors are unable to reproduce and only go through one Round of replication. Two components are required to produce such vectors. To the one requires a packaging cell that contains the MLV's gag, pole and env gene products by expressing psi-negative constructs so that these genes do not enter a retrovirus can be packed. The packaging signal of the retroviruses, which the controls efficient packaging of messenger RNA. On the other hand, a so-called Expression construct can be produced, which is a packaging in the retroviral vector and Transfer through the retrovirus allowed and that a coding and translatable region of the contains the desired gene product. Thus the expression construct must contain the packaging signal psi included. The gag, pol and env genes in the untreated packaging cell must be psi-negative so that corresponding messenger RNA does not enter the retrovirus particles is recorded. After transfer of the expression construct by transfection of the Appropriate vector DNA in the packaging cells are retroviral in the cell supernatant Delivered vector particles that contain only the expression construct, but not the psi-negative gag, pol and env genes so that these genes are not transferred to target cells.  

The tropism of retroviral vectors, i. H. the selection of the mammalian cells into which these Vectors that can transfer the expression construct are used by the env gene in the Packaging cell determined. The env gene is expressed in coat proteins, the transmembrane protein (TM) and translates the surface coat protein (SU), which is the outer coat of the retroviral Vector. The env gene products of the amphotropic MLV, which was previously mainly used for Gene transfer is used to allow gene transfer in a large number of different ways Mammalian cells. The amphotropic retroviral allows especially for the gene transfer into human cells However, vector does not selectively transfer genes into certain types of cells or tissues Humans or other mammals, because that is the arrival of amphotropic retroviral vectors and the Gene transfer mediating recipient protein (receptor) for the MLV coat proteins on the Surface of the mammalian cells can be found on almost all of these cells.

Gene therapy currently involves the stable transmission of different genes Cell culture, d. H. "ex vivo", in the foreground. Improvements to the retroviral vectors were made previously achieved by replacing the MLV's retroviral env gene with the env genes of other retroviruses were brought into the packaging cell. For example, instead of the MLV env gene env genes of the G protein of the "vesicular stomatitis virus (VSV)" (Burns et al., Proc. Natl. Acad. Sci. USA 90 (1993), 8033-8037). The resulting retroviral vectors showed below other increased stability. WO 96/17071 describes retroviral vectors which contain the env gene of the "human spumaretrovirus (HSRV)" instead of the MLV env gene. HSRV however, like amphotropic MLV, shows no specificity in cell infection. All so far The mammalian cells examined are permissive, regardless of the donor organism or the cell type for infection with HSRV (Schweizer et al, J Virol. 71 (1997), 4821-4824, and Russel et al., J Virol. 70: 217-222 (1996). The possible use of the env genes of the "simian sarcoma associated virus "(Takeuchi et al., Virology 186 (1992), 792-794), of the" feline leukemia virus, subgroup B "(Porter et al., Hum. Gene Ther. 7 (1996), 913-] 19), of the" feline endogenous virus RD114 "(Cosset et al., J Virol. 69 (1995), 7430-7436) and the" human T-cell leukemia virus I (HTLV-I) "(Vile et al., Virology 180 (1991), 420-424) was interpreted in certain experiments on. Attempts to produce retroviral vectors which contain the env gene of the lentiviruses HIV-1, HIV-2 or "simian immunodeficiency virus (SIV)" have so far not been successful. Such retroviral vectors would be the core proteins encoded by the MLV gag region, and the coat proteins encoded by the env region of another retrovirus, e.g. the HIV or SIV included.

To date, no vectors are available for selective gene transfer in CD4-positive mammalian cells Available. The present invention is therefore based on the object of retroviral To provide vectors in forms that are not the amphotropic receptor of mammalian cells, but target other receptors that are only expressed in certain tissues and cell types. These vectors are for specific gene delivery to selected mammalian cell types suitable. Another object is to develop a method for producing such retroviral vectors to develop.

According to the invention, these objects are achieved by the provision of retroviral vectors comprising virus nuclei and virus envelopes, which are characterized in that the virus nuclei come from murine leukemia virus (MLV) and the virus envelopes from human immunodeficiency viruses (HIV) or monkey immunodeficiency viruses (SIV). In particular, the retroviral vectors are characterized in that the virus envelopes from the human immunodeficiency virus 1 or 2 (HIV-1 or HIV-2) or from the monkey immunodeficiency virus [e.g. B. Cerecopithecus aethiops (SIVagm), Macaca mulatta (SIVmac), Pan troglodydytes (SIVcpz), Cerecopithecus mitis (SIVsyk), Papio sphinx (SIVmnd), Cercocebus atys (SIVsm) or Macaca nemestrina (SIVmne). Retroviral vectors whose virus envelopes contain the full-length surface protein and a shortened form of the transmembrane envelope protein are particularly preferred. Particularly preferred are retroviral vectors whose virus envelopes contain the full-length surface protein and a truncated form of the transmembrane envelope protein which has been extended by the C-terminus or any fragment of the transmembrane protein of the murine leukemia virus (MLV) or another retrovirus.

Furthermore, packaging cells are provided in which the psi-negative coat protein gene (env) of the lentiviruses HIV or SIV and the psi-negative gag / pol genes of the MLV are expressed will. These packaging cells also contain psi-positive expression constructs, which are transmitted using the retroviral vectors according to the invention.

In one embodiment of the invention, virus cores can be derived from a defined retrovirus originate in connection with expression constructs for the production of the retroviral Vectors are used because of their properties, here because of their existence of the packaging signal psi, into which virus nuclei are incorporated. The virus cores with the too  Transferring expression constructs are encased in foreign virus envelopes that are encoded by a different virus type or from another cell. The expression constructs are then transmitted using the retroviral vectors. The installation of the foreign virus envelope can e.g. B. by using the preferably shortened shape of the transmembrane Coat protein of the HIV-1 env gene pTr712 are mediated. Furthermore, the installation of third parties Virus envelope e.g. B. by using the truncated form of the transmembrane coat protein of SIVagm3 env gene Δ0env can be conveyed. In a further embodiment of the invention can do full length transmembrane proteins or modified transmembrane proteins Append the C-terminus of the transmembrane envelope protein of the MLV or the C-termini of the transmembrane envelope proteins other viruses to the truncated or to the full-length trans membrane proteins can be used. These modified coat proteins can be in vectors be included. MLV-derived ones are particularly preferred for such vectors Capsid particles which contain the coat proteins of other retroviruses, in particular other lentiviruses such as HIV or SIV included. These vectors infect the desired cell type via the cellular one Receptor of the virus from which the foreign envelope is derived.

In a further embodiment according to the invention, any cell with a psi negative expression gene for gag and pol genes transfected. Furthermore, the cell with a Expression construct comprising a psi packaging signal and which is delivered to a target cell transferring genetic information to be transfected. The cell is then joined with another Expression gene transfected that contains the genetic information for foreign coat proteins. The so The cell line produced produces retroviral vectors which are the genetic to be transferred Contain information.

In a preferred embodiment, the MLV env negative packaging cell line TELCeB6 (Cosset et al., J Virol. 69 (1995), 7430-7436) with the truncated HIV-1 env gene pTr712 of plasmid pLβAc / env-Tr712-neo (Wilk et al., Virology. 189 (1992), 167-177; Krausslich et al., Virology 192 (1993), 605-617). This creates one Packaging cell line which contains the retroviral vectors of the type MLV (HIV-1) according to the invention releases the cell culture supernatant. These vectors contain capsid particles, which due to the Expression of the gag / pol gene of the MLV arise in the cell, and envelope proteins of the HIV-1, which is due to the intracellular expression of the shortened version Tr712 of the HIV env gene in the vector particles are recorded. The vectors according to the invention contain this  HIV-1's full-length surface protein gp120-SU and the truncated form of the transmembrane Coat protein, the coding portion of which is due to a stop codon (position 712). in the Individuals have been shown to use this to produce retroviral vectors for the selective Gene transfer to CD4-positive mammalian cells resulted. CD4-positive, but not CD4-negative cells two otherwise identical lines could be transduced specifically with the vectors, i. H. the expression construct gene was transferred. The transduction was carried out in the presence of Antibodies that also inhibit HIV infection are inhibited. The Surface coat protein of HIV was produced on the surface of the gene transfer vectors proven.

In a further preferred embodiment, the MLV env negative packaging cell line TELCeB6 transfected with the shortened SIVagm3 env gene Δ0env. The received The packaging cell line secretes the retroviral vectors of the type according to the invention MLV (SIVagm3) in the cell culture supernatant. These vectors also contain capsid particles of the MLV and coat proteins of the SIVagm3, which are characterized by the intracellular expression of the shortened version Δ0env of the SIVagm3 env gene can be included in the vector particles.

The present invention thus opens up the following possibilities:

• - selectively transfer genes into CD4-positive mammalian cells,
• - Further efficiency increases in gene transfer by improving the env gene constructs in to achieve comparable packaging cells,
• - To develop gene therapy strategies for which a selective gene transfer into CD4-positive Mammalian cells appear necessary or helpful,
• - In particular gene therapy strategies for the treatment or prophylaxis of HIV infection of the To develop humans, whereby HIV-inhibiting genes, e.g. B. antisense genes, RNA bait genes or transdominant-negative mutant genes of HIV or other lentiviruses in CD4-positive Cells are transferred
• - In particular, gene therapy strategies for the introduction of genes into CD4-positive cells develop, for the treatment or prophylaxis of hereditary diseases, such as. B. ADA deficiency or other genetically treatable diseases where the introduction to certain Cells is beneficial, and
• - to examine in detail the entry of lentiviruses into mammalian cells.

The illustrations serve to explain the invention.

Fig. 1 shows schematically the principle of gene transfer using retroviral vectors in mammalian cells.

Fig. 2 shows a schematic of the production of a retroviral vector (taken from "Molecular Biotechnology", Principles and Applications of Recombinant DNA, BR Glick and JJ Pasternak, ASM Press, Washington, DC, 1994, page 411, and translated into German).

Fig. 3 shows schematically a generally applicable production route for retroviral vectors (retroviral transfer vectors) by transfection of an initially expression construct negative packaging cell with an expression construct which, due to the presence of the packaging signal (here: psi), the gene (therapeutic gene sequence) to be transferred into the retroviral vectors smuggled in.

Fig. 4 shows a special production route for retroviral vectors of the type MLV (HIV-1). The MLV gag / pol expression gene and the psi-positive expression construct pMFGInsLacZ are shown, which are expressed by the MLV env-negative packaging cell TELCeB6. The env gene variant of HIV-1 (Tr712), which contains the regions coding for the full-length surface coat protein gp120-SU and the shortened version of the transmembrane coat protein (Agp41-TM), is used in the production of the MLV (HIV-1) vectors also expressed in this cell.

Fig. 5 shows the general structure of retroviral vectors, which contain the virus core of a certain virus in combination with the virus envelope of another virus, explained using the example of the MLV (HIV-1) vectors.

Fig. 6 shows schematically the cloning scheme for the cloning strategy of the SIVagm3 env expression construct pRep wt env and the shortened env genes, explained using the example of the variant Δ0 env.

Fig. 7 shows schematically the cloning scheme for the cloning strategy of the SIVagm3 env fusion genes Δ0MLV env and Δ7MLV env.

Fig. 8 shows the nucleic acid sequences of the oligonucleotides used to clone the SIVagm3 env expression constructs. The nucleic acid sequences of the restriction sites are underlined.

Fig. 9 shows schematically the transcriptional units of the SIVagm3 env constructs.

Fig. 10 shows the amino acid sequences of the intracellular domains of the gene products of SIVagm3 env constructs. The sequences of SIVagm3 and MLV are given for comparison. The amino acids are shown in the one-letter code. Amino acids derived from MLV sequences are underlined. The numbers contained in the names of the constructs relate to the N-terminally remaining amino acids after the transmembrane region and before the stop codons inserted by means of recombinant PCR. However, insertion of a Not I restriction site results in two or three amino acids, which do not occur in the native sequence of SIVagm3. The amino acids encoded by the inserted Not I cut are shown in bold. "..." stands for amino acids of SIVagm3, which are not listed in detail. "*" denotes the C-terminus of the proteins. The length of the intracellular domains is indicated. "aa" means amino acids. "TMR" means transmembrane region. The term "MLV" stands for the inserted 3 'lying sequences of the MLV env gene. The inserted C-termini of the MLV each contain the so-called p2 protein (consisting of 16 aa), which is cleaved intracellularly by proteolysis before the coat proteins are incorporated into the virions.

Fig. 11 is a graphical representation of the efficiency of the formation of MLV (SIVagm) vectors after transfection of the env variants in TELCeB6 / rev cells. The T cells were cocultivated in the same medium and in the presence of the transfectants for two days and then examined for the gene transfer by means of the X-Gal test. The values given represent mean values from two experiments. "Mock" denotes the negative control.

Fig. 12 is a graphical representation of the results of the titration of the MLV (SIVagm) vector stocks on different CD4⁺ cell lines.

Figure 13 is a graphical representation of the results of inhibition of T cell transduction by blocking the cellular CD4 receptor by monoclonal anti-CD4 antibodies. The concentrations of the antibody IOT4a used are given in the legend. The transduction efficiencies achieved in the absence of the antibodies were equated as a reference 100% and the titers in the presence of the antibodies were expressed as a percentage of this positive control.

The term retroviral vector used here means a replication-deficient retroviral Virus particles which, instead of the retroviral mRNA, introduce a foreign RNA from a gene, e.g. B. a therapeutic gene or its fragment or a reporter gene.

The term "therapeutic gene" as used herein means a nucleic acid sequence that can be found in the Target cell is to be introduced by means of the retroviral vector and comprises complete genes, their fragments, antisense nucleic acids and the like. The term SIV used here means viruses of the monkey immunodeficiency virus family, e.g. B. Cerecopithecus aethiops (SIVagm), Macaca mulatta (SIVmac), Pan troglodydytes (SIVcpz), Cerecopitheeus mitis (SIVsyk), Papio sphinx (SIVmnd), Cercocebus atys (SIVsm) or Macaca nemestrina (SIVmne). The term SIVagm3 used here denotes the molecular clone SIVagm3mc (Baier et al., J: Virol. 62 (1989), 4123-4128).

The production of MLV (HIV-1) and MLV (SIVagm3) vectors of the invention will explained in more detail below.

First, a DNA sequence is created, which is the formation of the necessary proteins induced that are able to form virus nuclei. The DNA sequence is divided into a human host cell transferred and expressed there. The DNA sequence also contains Operator elements necessary for the expression of the DNA sequence, which are the formation of the virus nuclei. A second DNA sequence is placed in the host cell obtained in this way which leads to the formation of coat proteins which are not derived from the virus from which the virus nuclei come from. Another DNA sequence is then introduced, which is from the viral nucleus is packaged and contains sequences that form the therapeutic Gene sequences or the desired proteins in the cell result in using the retroviral Vector therapeutic genes are to be transferred.  

The DNA sequence which leads to the expression of the vector envelope proteins can preferably be from env gene of the HIV-1, HIV-2 or other HIV strains, the SIV, e.g. B. Cerecopitheczis aethiops (SIVagm), Macaca mulatta (SIVmac), Pan troglodydytes (SIVcpz), Cerecopithecus mitis (SIVsyk), Papio sphinx (SIVmnd), Cercocebus as (SIVsm) or Macaca nemestrina (SIVmne) come. The env genes used can be the original version of the viruses mentioned correspond to or shortened forms of these env genes or even modified forms of these env genes be. The particularly preferred HIV-1 env gene pTr712 leads to the formation of a full length upper surface protein gp120-SU and a truncated transmembrane protein. That too particularly preferred SIVagm3 env gene Δ0env leads to the formation of a full length upper surface protein gp130-SU and a truncated transmembrane protein. The Transmembrane protein can also e.g. B. by attaching the C-terminal domain or one any other fragment of the transmembrane protein of the MLV or another virus be modified. For example, when using MLV capsid particles, the C-terminus of the transmembrane protein of the MLV instead of the C-terminus of the HIV-1 env gene or SIVagm env gene can be used. The cleavage site of the C-terminal p2 peptide can be modified be or not.

The viral vectors according to the invention mentioned can be used to transmit therapeutic Genes can be used in certain cell types. For example, the MLV (HIV-1) and MLV (SIVagm3) vectors genes according to the invention specifically in CD4-positive Cells. These vectors thus have the tropism of HIV-1 or SIVagm3, their Coat proteins they contain.

The following examples illustrate the invention and are not to be understood as restrictive.

example 1 Production of MLV (HIV-1) vectors 1.1 cell lines and plasmids

All plasmids used were from transformed E. coli of the known strains DH10α or HB101 prepared. The molecular cloning of the expression constructs pLβAc / env-neo is von Krausslich et al., Virology 192 (1993), 605-617 and pLssAc / env-Tr712-neo is from Krausslich et al., Virology 192 (1993), 605-617 and Wilk et al., Virology. 189: 167-177 (1992)  described. These expression constructs encode the HIV-1 env gene variants and that Neomycin resistance gene. The expression construct pCRUCA, which the env gene of amphotropic MLV has been described by Wilk et al., Virology. 189: 167-177 (1992) and Battini et al., J Virol. 66: 1468-1475 (1992). The TELCeB6 packaging cell line that does that retroviral expression construct MFG-nlsLacZ and the genes gag and Pol of the MLV are expressed, was published by Cosset et al., J Virol. 69: 7430-7436 (1995). The HeLa-CD4⁺ cells were obtained from the MRC AIDS Program Reagents Project, the 293 cells were from ATCC (ATCC CRL 1573) related. All adherent cells were modified in Dulbecco's Eagles Medium (GIBCO / BRL, Eggenstein, Germany) in the presence of 10% fetal Calf serum kept. The Molt 4 human AT cell line (ATCC CRL 1582) was developed in RPMI-1640- Medium propagated with 10% fetal calf serum (GIBCO / BRL, Eggenstein, Germany). The Transfection of the packaging cell line TELCeB6 with the expression construct pTr712 was carried out using lipofectamine (GIBCO / BRL, Eggenstein, Germany) according to the information provided by the Manufacturer carried out. After transfection of the plasmid pREP4 (Invitrogen, Leek, Netherlands) the hygromycin selection in the presence of 200 mg / ml hygromycin B (Sigma, Deisenhofen, Germany). One of the resulting clones represents the Cell line TELCeB6 / pTr712-K14.

1.2 Viral infection, determination of titers and neutralization experiments

The adherent cells were seeded in 24-well (well) plates at a density of 4 × 10 ⁴ cells per well or in 6-well plates at a density of 2 × 10 ⁵ cells per well. The Molt-4 cells were seeded in 6-well plates with a density of 8 × 10 ⁵ . Before infection, the cells were incubated in cell medium overnight. For infection, the target cells were co-incubated for 3 hours with 1 ml of supernatants containing diluted or undiluted retrovirus particles. The supernatants containing virions were previously passed through a 0.45 mm filter to remove the contaminating cells. Two days after infection, the target cells were examined for β-Gal expression using the x-Gal test. The viral titers were determined as described. The titers are given in colony forming units per ml (colony fonning units, cfu). Serum from an HIV-1 infected donor was used to neutralize the pseudotyped vectors.

1.3 Immunostaining of the transfectants

The TELCeB6 cells transfected with the DNA of the plasmids encoding the envelope proteins of the HIV-1 were washed with PBS and then incubated for 15 min with ice-cold methanol. After repeated washing, blocking buffer (PBS / 2% BSA) was added to the cells for one hour. The cells were washed again and then incubated with a 1: 1000 diluted anti-HIV-1 serum solution for 1 hour. After repeated washing, the cells were incubated with peroxidase-coupled protein G (Bio-Rad, Krefeld, Germany). Finally, the antigen-presenting cells were stained by adding substrate buffer (H ₂ O ₂ with 3-amino-9-ethylcarbazol, Sigma, Deisenhofen, Germany). It could be shown that the plasmid pTr712 allowed the formation of the envelope proteins derived from HIV-1 in the transfectants.

1.4 Western blot analysis

The cell lysates were prepared and the Western blots were carried out in the usual manner Method. The virus particles in the supernatants are those derived from HIV-1 Envelope proteins encoding plasmids were transfected using packaging cells Ultracentrifugation (45 min at 200,000 × g and 40 ° C) concentrated. The centrifugates were taken up in sample buffer and separated by means of SDS polyacrylamide gel electrophoresis. Western blot analysis was done using a goat serum against HIV-1 gp120-SU and peroxidase-coupled Protein G. The protein bands were detected using an ECL detection kit (Amersham, Braunschweig, Germany). As well in the cell lysates as well as in the vector particles, the surface coat protein gp120-SU des HIV-1 can be detected.

1.5 Membrane fusion property of the HIV-1 coat protein

A subconfluent culture of the packaging cell line TELCeB6 / pTr712-K14 was carried out with Jurkat-Zel len overlaid, propagated for 48 hours and then photographed. It was a series of Syncytia observed so that the functionality of that formed by the above cell line Envelope proteins of HIV-1 could be shown.  

Example 2 Production of MLV (SIVagm3) vectors 2.1 Cloning the SIVagm3 env expression constructs

The plasmid pRep 4 was used to clone recombinant variants of the SIVagm3 env gene (Invitrogen, Leek, The Netherlands). The first step was to produce the env variants the sequence between bases 5713 and 8411 of SIVagm3 from plasmid pMB2 (Baier et al., J Virol. 63 (1989), 5119-5123) by means of recombinant PCR (rPCR) using the Oligonucleotides near AGM ENV + (SEQ ID NO: 1) and Xho AGM ENV- (SEQ ID NO: 2) amplified and inserted into the vector pRep4 via the restriction sites Nhe I and Xho I. The cDNA produced by rPCR comprises both rev exons and the entire reading frame of the env gene. The plasmid obtained was called pRep wt env and subsequently served as a template for further rPCRs and as a vector for further cloning.

First, the shortened env genes Δ0 env, Δ7 env and Δ16 env were cloned. To do this first the respective oligonucleotides Not STOP 0 - / + (0-: SEQ ID NO: 3, 0+: SEQ ID NO: 4), Not STOP 7 - / + (7-: SEQ ID NO: 5, 7+: SEQ ID NO: 6) and Not STOP16 - / + (16-: SEQ ID NO: 7, 16+: SEQ ID NO: 8), used in PCRs to set the desired stop codon and a 5 ' Advertise Not I restriction interface. The respective flanking oligonucleotides were used Oligonucleotides SIV ENV HIII + (SEQ ID NO: 9) and Xho AGM ENV- (SEQ ID NO: 2) to the Amplificates as small as possible and thus the probability of incorrect amplification to be kept low by the tag polymerase. The amplificates obtained in the individual PCRs were isolated in the usual way and then used in fusion PCR (amplificate SEQ ID NO: 9/3 with amplificate SEQ ID NO: 4/2; Amplificate SEQ ID NO: 9/5 with amplificate SEQ ID NO: 6/2; Amplificate SEQ ID NO: 9/7 with amplificate SEQ ID NO: 8/2). The first amplificate contains the sequences of the SIVagm env from the Hind III interface (6817) to the Primer (Not STOP 0- / 7- / 16-), which has a NOT I interface and the one to be inserted Stop codon as well as the 3 'following sequences in the template. The second amplificate starts 5 'with the + primers (Not STOP 0 + / 7 + / 16 +), which are the last 3'-lying bases of the contains first amplificates, a Not I interface and the stop codon, and ends with the Sequence of the Xho AGM ENV primer. The amplificates used in the fusion PCR thus had overlapping sequences that allowed hybridization. Using fusion PCR the amplified products hybridized to one another using the primers Xho AGM ENV-  and SIV ENV HIII + amplified. The resulting fusion fragments were after restriction (Hind III / Xho I) cloned into the vector Rep wt env (Hind III / Xho I). The env variants Δ0MLV env and Δ7MLV env were produced from the variants Δ0env and Δ7env. For this were the 3 'lying regions of the MLV env gene, which the intracellular portions of the Encode TM protein p15 by means of rPCR using the oligonucleotides MLV emergency (SEQ ID NO: 10) and MLV Not 7+ (SEQ ID NO: 11) or MLV Not 0+ (SEQ ID NO: 12) amplified. Molecular clone pKA1558 was developed by Scov et al., J Gen. Virol. 74: 707-714 (1993) described and served here as a matrix. The amplificates were obtained from the Restriction interface Not I inserted in the env variants Δ0env and Δ7env.

2.2 polymerase chain reaction (PCR)

The Taq DNA polymerase from Perkin-Elmer, Langen, Germany was used to amplify the desired DNA sections. A standard PCR (100 μl mixture) contained: 1 × PCR buffer (10 mM Tris / HCl, pH 8.8, 50 mM KCl, 1.5 mM MgCl ₂ , 0.01% gelatin), 10 amplificate SEQ ID NO: 9/7 with amplificate SEQ ID NO: 8/2 µM per oligodeoxynucleotide primer, 200 µM per deoxynucleotide (dNTP), 2.5 units Taq polymerase and 100 ng plasmid DNA. The PCR program for the primer pairs, amplificate SEQ ID NO: 9/3 with amplificate SEQ ID NO: 4/2, amplificate SEQ ID NO: 9/5 with amplificate SEQ ID NO: 6/2 and amplificate SEQ ID NO: 9 / 7 with amplificate SEQ ID NO: 8/2 was as follows:

• 1. 94 ° C 300 sec.
• 2. 94 ° C 45 sec.
• 3. 55 ° C 120 sec.
• 4. 72 ° C 180 + 2sec.
• 5. 10 cycles 2-4.
• 6. 94 ° C 45 sec.
• 7. 60 ° C 120 sec.
• 8. 72 ° C 180 + 2 sec.
• 9. 10 cycles 6-8.
• 10. 72 ° C 600 sec.

2.3 Cell lines, media, transformation and transfection

Plasmids were introduced into the E. coli strains DH5α, DH10B, Top10F '(GIBCO / BRL, Eggenstein, Germany) and GM 2163 (Invitrogen, Leek, The Netherlands) in a conventional manner. The transfection of the packaging cell line TELCeB6 or TELCeB6 / rev with the SIVagm env expression constructs was carried out using lipofectamine (GIBCO / BRL, Eggenstein, Germany) according to the manufacturer's instructions. The TELCeB6 packaging cell line, which expresses the retroviral expression construct MFG-nlsLacZ and the gag and pol genes of the MLV, was described by Cosset et al., J Virol. 69: 7430-7436 (1995). A plasmid (pCMV-rev, NIH Research and Reference Reagent Program, catalog number 1443) was introduced into the cell line TELCeB6 / rev by stable transfection, which allowed the expression of the rev gene of HIV-1 under the transcriptional control of a CMV promoter. The TELCeB6 / rev cell line thus additionally expresses the rev gene of HIV-1. The cell lines TELCeB6 and TELCeB6 / rev were cultivated in the media described below for adherent cells with the addition of glucose (4 g / L). Adherent HeLa-CD4 + cells were obtained from the MRC AIDS Program Reagents Project, the HeLa cells from ATCC (CCL 2). All adherent cells were in Dulbecco's Modified Eagles Medium (GIBCO / BRL, Eggenstein, Germany) in the presence of 10% complement-inactivated fetal calf serum (FKS; Biochrom KG, Berlin, Germany), 2 mM L-glutamine (Biochrom KG, Berlin, Germany) and antibiotics (0.1 mg / ml nystatin, 100 units / ml penicillin, 50 mg / ml streptomycin; Biochrom KG, Berlin, Germany) at 37 ° C, 5% CO ₂ and saturated water vapor atmosphere in a cell incubator (Cytopertn, Heraeus, Germany). The suspension cells C8166 (human leukemia T cells which contain the defective HTLV-1 genome (NIH Research and Reference Reagent Program, catalog number 404), Jurkat cells (human leukemia T cells; NIH Research and Reference Reagent Program, Catalog number 177) and Molt4.8 cells (human leukemia T cells; NIH Research and Reference Reagent Program, catalog number 175) were in RPMI 1640 (GIBCO / BRL, Eggenstein, Germany) with 10% FCS, 2 mM L-glutamine (Biochrom KG, Berlin, Germany) and antibiotics (0.1 mg / ml nystatin, 100 units / ml penicillin, 50 mg / ml streptomycin; Biochrom KG, Berlin, Germany).

2.4 Immunoperoxidase Antiperoxidase Test (IPAP)

In order to check the correct expression of the env variants, all env constructs were transfected in TELCeB6 cells. Two days after the transfection, it was tested by means of IPAP whether the transfectants expressed the respective env variant. The TELCeB6 cells were washed in 35 mm cell culture dishes with PBS and fixed with -20 ° C cold methanol at -20 ° C for 15 minutes. The non-specific binding sites on the cells washed again with PBS were then blocked using a blocking buffer (PBS + 2% BSA) for one hour at room temperature (RT). The cells were then incubated for one hour with a SIVagm antiserum (from a pig monkey (Macaca nemestrina) infected with SIVagm in the usual manner and diluted with blocking buffer) at 37 ° C. After further washing with PBS, the Fc portions became the primary Antibodies were detected using horseradish peroxidase-conjugated protein G (BIO-RAD, Munich, Germany) after incubation for one hour at 37 ° C. After two washing steps with PBS, the substrate solution (4 mg of 3-amino-9-ethylcarbazole in 1 ml of dimethylformamide) was added dissolved, 19 ml 20 mM NaOAc buffer, pH 5 and 30 µl H ₂ O ₂ ; filtered through a 45 µm filter.) After a few minutes of incubation at RT, the cells expressing virus protein showed a clear red color. Except for the plasmids pRep Δ7env and pRep Δ7MLVenv, all constructs allowed expression of the envelope proteins of SIVagm 3. When analyzing the sequencing data, it was found that the two variants pRep Δ7env and pRep Δ7MLVenv had a stop codon in the second rev exon, which was created by inserting the Not I interface. The Rev protein, which was formed incompletely, was apparently unable to ensure the transport of the mRNA transcripts of the env genes from the cell nucleus.

2.5 induction of syncytia

Since the analysis using IPAP only detects the viral coat proteins formed, these also had to be checked for functionality. By means of syncytia induction it should be shown that the coat proteins are presented on the cell membrane and induce syncytia in CD4⁺ cells can. For this purpose, the SIVagm env constructs were transfected in TELCeB6 / rev cells. At the the following day, the transfectants were overlaid with cells from the T cell line Molt4.8 and 24 h photographed later. The SIVagm-Hüll presented on the cell membrane of the packaging cells Proteins are able to convert the membrane-bound CD4 proteins of the T cells  tie. This leads to the fusion of the two cell types. Differentiate these syncytia differ in size from the parental cells. All tested env variants induced syncytia in this test, however, the extent of syncytia induction was very different. The variants Δ0env and Δ16env induced large syncytia, while the other variants including the wt env produced only weak syncytia induction. However, could be shown for all variants that their expression led to the formation of membrane-bound proteins, which are able were to bind CD4.

2.6 Detection of β-galactosidase activity (X-Gal test)

Using the X-Gal test, a successful transduction of human cells with the MLV (SIVagm) derived vectors was demonstrated based on the successful transfer of the packable construct MFGlnsLacZ. The detection of the β-galactosidase activity in transduced cells was carried out according to a modified method (Sanes et al., EMBO J. 5 (1986), 3133-3142) by means of X-Gal staining. In addition to the β-galactosidase substrate (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside; Sigma, Deisenhofen, Germany) [1 mg / ml], the reaction mixture contained 5 mM potassium ferricyanide (Sigma, Deisenhofen, Germany ), 5 mM potassium ferrocyanide (Sigma, Deisenhofen, Germany) and 2 mM MgCl ₂ in PBS. The cells washed with PBS were fixed with PBS, 2% formaldehyde and 0.2% glutardialdehyde for 10 min at room temperature. The cells were then washed with PBS and incubated with the X-Gal reaction mixture at 37 ° C. for 5 to 24 hours. The intracellular blue staining was able to detect the β-galactosidase.

2.7 Transduction of T cells with MLV (SIVagm3) vectors

4 × 10 ⁵ TELCeB6 / rev cells were sown in cell culture dishes with a diameter of 35 mm and transfected with the SIVagm env constructs 24 hours later. The media of the transfectants were renewed the following day. Then cocultivation vessels (Costar, Cambridge, USA) were inserted into the culture dishes of the packaging cells, which allow the free exchange of media and the vectors contained therein, but prevent direct cell-cell contact. In each of these inserts, 10 ⁶ T cells of the Molt4.8 line were sown and cultivated together with the transfectants for two days.

The T cells were then expanded for a further two days and finally using X-Gal Test for the expression of the reporter gene lacZ was examined.

Transfection of all SIVagm env variants with an intracellular domain no longer than 19 Amino acids led to the formation of pseudotyped MLV vectors, which are the T cells successfully transduced. The variants wt env and A36env did not produce any measurable quantities of these vectors. The plasmid pHIT456 (Soneoka et al., Nucl. Acid. Research 23 (1995), 628-633), which encodes the env gene of the amphotropic MLV.

Example 3 3.1 Establishment of stable transfected packaging cell clones

To produce high-titer vector stocks, TELCeB6 cells were used with the corresponding env constructs stably transfected and then selected. The selection media corresponded to the media used for cultivation, but additionally contained the neomycin Analog G418 (800 µg / ml) for the selection of neo⁺ cells (HIV-1 env constructs) or Hygromycin B (200 µg / ml) for the selection of hyg⁺ cells (SIVagm env constructs). The Antibiotics were purchased from Sigma (Deisenhofen, Germany). The selection of transfected Cells started two days after transfection and continued until approximately ten days Cell colonies had formed from single cell clones. The clonal cells were Eppendorf pipette detached from the culture dish and resuspended. These single cell clones were first expanded into 24-well plates and based on the formation of vectors by titration suitable target cells examined.

3.2 Generation of pseudotyped MLV vectors

The vectors produced by the packaging cells were prepared as follows: The media confluent packaging cell cultures in large cell culture bottles (800 ml) were removed. The cells were coated with 15 ml of fresh medium and cultured overnight. Subsequently the supernatants were filtered "cell-free" through a 0.45 µm syringe filter and either directly used for transduction or stored in liquid nitrogen.  

3.3 Determination of the vector titers

To determine the vector quantities in supernatants of the packaging cells, these were used in various dilutions with fresh culture medium in a total volume of 1 ml for the transduction of permissive target cells (Molt4.8, C 8166, Jurkat, HeLaCD4⁺). The selected dilutions were 1: 1, 1:10, 1: 100, 1: 1000. The adherent target cells HeLa CD4⁺ were sown at a density of 2 × 10 ⁵ cells per cell culture dish (35 mm diameter) one day before the start of transduction. Before the vector-containing media were added, the cells were washed with PBS and incubated for two hours at 37 ° C. in the presence of the vectors. The supernatants were then removed, the cells were washed again and then expanded for a further two days before the transduced cells were stained using the X-Gal test. Suspension cells were supplied with fresh media one day before the start of transduction. For the transduction, 10 ⁶ cells were pelleted in a conventional manner (centrifuged) and resuspended directly in the vector-containing dilutions. After two hours, the cells were washed and, after two days of expansion, also examined for the gene transfer by means of an X-Gal test. The LacZ⁺ cells stained in this way were counted in 10 to 20 fields of view using an optical microscope (Axiovert 35, Carl Zeiss, Jena) and extrapolated to the total area of the culture dish. The dilution of the vector-containing supernatants was taken into account and the titer given in cfu / ml. Molt4.8 cells gave the best transduction efficiencies, whereas the Jurkat and C8166 T cell lines were much more difficult to transduce. The HeLaCD4⁺ cells seem to be hardly permissive for the MLV (SIVagm) vectors. The MLV (HJV-1) vectors, however, were able to efficiently transduce all tested CD4 getest cell lines.

Example 4 Detection of CD4-dependent transduction using MLV (SIVagm) vectors

The T cell line Molt4.8 was held 30 minutes before the addition of the MLV (SIVagm3) vectors in media containing different concentrations (0 µg / ml, 0.625 µg / ml, 1.25 µg / ml, 2.5 µg / ml, 5 µg / ml) of the CD4-binding monoclonal antibody IOT4a (company dianova, Hamburg) contained. The antibody inhibits the entry of various HIV and SIV isolates by blocking the cellular receptor CD4; see. Sattentau et al. (1986) Science 234: 1120-1127. The T cells were then transduced using the MLV (SIVagm) vectors in the presence of the above-mentioned concentrations of the anti-CD4 antibody IOT4a. The cells were then washed and examined two days later for the gene transfer by means of the X-Gal test. As a positive control, target cells were transduced in the absence of the antibodies and selected as a reference (100%). There was a clearly concentration-dependent correlation of the efficiency of the gene transfer with the amount of antibodies used.

Claims (14)

1. Retroviral vectors comprising virus nuclei and from surface envelope proteins and transmembrane envelope proteins existing virus envelopes, the virus nuclei from murine leukemia virus (MLV) and the viral envelopes of human Immunodeficiency viruses (HIV) or monkey immunodeficiency viruses (SIV). 2. Retroviral vectors according to claim 1, wherein the virus envelopes of human immunodeficiency virus 1 or 2 (HIV-1 or HIV-2) or monkey immunodeficiency virus Cerecopithecus aethiops (SIVagm) or Macaca mulatta (SIVmac) or Pan troglodydytes (SIVcpz) or Cerecopithecus mitis (SIVsyk) or Papio sphinx (SIVmnd) or Cercocebus as (SIVsm) or Macaca nemestrina (SIVmne). 3. Retroviral vectors according to claim 1 or 2, wherein the virus envelopes the full length upper surface protein and an unabridged or abbreviated form of the transmembrane Envelope protein included. 4. retroviral vectors according to claim 1 or 2, wherein the virus envelopes the full length upper surface protein and a truncated form of the transmembrane envelope protein. 5. The retroviral vectors of claim 4, wherein the virus envelopes are full-length surface protein and a shortened form of the transmembrane envelope protein, that by the C-terminus or any fragment of the transmembrane protein of murine leukemia virus (MLV) or other retrovirus has been extended. 6. Process for the production of packaging cells that contain retroviral vectors of claims 1 to 5, comprising transfecting a packaging cell (gag, pol and expression construct positive), which produces coat proteins derived from the MLV or not produced (env-negative), with an expression gene that is genetic Contains information for coat proteins (env-positive). 7. Process for the production of packaging cells that contain retroviral vectors of claims 1 to 5, comprising transfecting a cell with  Expression genes for gag and pol, and / or an expression construct, comprising a Packaging signal and the genetic information to be transferred, and a Expression gene that contains the genetic information for coat proteins. 8. The method according to claim 6, wherein the cell line as MLV-env-negative packaging cell TELCeB6 is used. 9. The method according to any one of claims 6 to 8, wherein as env expression gene pLβAc / env-Tr712-neo or pRep Δ16 env, pRep Δ7 env, pRep Δ0 env, pRep Δ7MLV env or pRep Δ0MLV env is used. 10. packaging cells, obtainable by the method according to any one of claims 6 to 9. 11. Use of the retroviral vectors according to one of claims 1 to 5 as medical tools (medicines). 12. Use of the retroviral vectors according to one of claims 1 to 5 for the preparation a drug used to deliver genes to CD4-positive mammalian cells. 13. Use of the retroviral vectors according to one of claims 1 to 5 for the preparation of a drug for the transfer of genes into CD4-positive cells in humans. 14. Use of the retroviral vectors according to one of claims 1 to 5 for the preparation a drug for the genetic modification of CD4-positive cells or for Provision of an active ingredient in the context of gene therapy.