Abstract:
The invention provides a viral self-inactivating (SIN) vector on the basis of the avian sarcoma leukosis virus (ASLV) as well as a split-packaging system comprising in addition to the SIN vector a first helper plasmid serving for the expression of the viral fusion protein gag-pol and a second helper plasmid serving for the expression of the retroviral envelope protein (env). The first and second helper plasmid, for example contained in a packaging cell line or transiently transfected, serve for the generation of non-replicating (RCR-incompetent) viral particles containing RNA having a SIN LTR according to the invention at the 3′ terminus, wherein the RNA can have a therapeutically effective section which e.g. is denoted a transgene. This 3′ SIN LTR contains an extensive deletion of the U3 region which in the course of the reverse transcription is copied into the 5′ LTR. In addition, in the SIN vector all coding regions of ASLV as well as the retroviral splice donor site are removed.

Description:
The present invention relates to a viral self-inactivating (SIN) vector on the basis of the avian sarcoma leukosis virus (ASLV), to viral particles containing the vector according to the invention, to a split-packaging system comprising or consisting of the SIN vector, a first helper plasmid serving for the expression of the viral fusion protein gag-pol and a second helper plasmid serving for the expression of the retroviral envelope protein (env), as well as to a method for the production of the viral particle by means of the split-packaging system, especially by expression in cultured human cells. Furthermore, the invention relates to the viral particle for use as medicament, especially for transfer of a transgene into cells, as well as to the use of the viral particle for the production of the medicament. The first and second helper plasmid, for example contained in a packaging cell line or transiently transfected, serve for the generation of nonreplicating (RCR-incompetent) viral particles containing RNA having a SIN LTR according to the invention at the 3′ terminus, wherein the RNA can have a therapeutically effective section which e.g. is denoted a transgene. This 3′ SIN LTR comprises an extensive deletion of the U3 region which in the course of the reverse transcription is copied into the 5′ LTR. 
     For the expression of a transgene, which e.g. for use for the gene therapy can be a human or heterologous gene, the viral SIN vector preferably contains an expression cassette arranged in 5′ to the 3′ SIN LTR. 
     The vector according to the invention also in human packaging cells allows the generation of viral particles at a high titre and is characterized in that during the transduction of target cells, especially of human cells, by contacting the cells extracorporeally, i.e. in vitro, or by means of administration of a particle adapted to this purpose to a patient cells transduced in vivo are generated expressing the transgene in a stable way and with low reduction, respectively, over a long period of time, wherein the frequency of the transformation of target cells by oncogene activation is low. The expression cassette has a promoter in 5′ to the nucleic acid sequence encoding the transgene and a post-transcriptional regulatory element (PRE), especially a WPRE (PRE of the woodchuck hepatitis virus (WHV)), preferably in 3′ to the sequence encoding the transgene. 
     STATE OF THE ART 
     Hughes (Folia Biologika 50, 107-119 (2004)) describes a series of viral vectors on the basis of the avian leukosis virus (ALV) which for better production of replication-competent retroviruses (RCR) have a functioning LTR. (RCAS: Replication-Competent ASLV LTR with a Splice Acceptor). In this system all components for the virus production lie on one plasmid (as in the full-length virus), and a possible transgene is inserted into the 3′ untranslated region (UTR). 
     Hu et al. (Molecular Therapy, 1617-1623 (2008)) show that ASLV is not prone to preferably insert inside or in the proximity of proto-oncogenes and that the LTRs of ALSV do not tend to activate adjacent genes and therefore conclude that ALSV should form a suitable basis for viral vectors. It is indicated that a deletion of the enhancer region (enhancer) in the LTR or the inhibition of through-transcribing over a termination signal could be achieved by an improved polyadenylation. 
     Hu et al. (Human Gene Therapy 691-700 (2007)) and Mitchell at al. (Plos Biology, e234, 2004) show that ALSV is not prone to preferably insert inside or in the proximity of proto-oncogenes, especially without a clear preference for regions close to the promoter. Accordingly, the danger of insertional mutagenesis in comparison to the clinically used vector systems from murine leukemic virus (MLV) and human immunodeficiency virus (HIV) is estimated as lower. It is indicated that a deletion of the enhancer region (enhancer) in the LTR or the prevention of through-transcribing over a termination signal would be desirable. Due to the vector architecture (1 plasmid, replicating) this system is usually not suitable for clinical applications. 
     Hu et al. show that viral vectors derived from ASLV having intact LTRs do not replicate in mammalian cells. The exact reason for this is unclear. 
     Due to the retroviral architecture of 2 LTRs retroviruses have intrinsically weak polyA signals. For lentiviral vectors it is known that the through-transcribing from the 3′ terminus of the inserted viral vector can lead to undesired gene activation according to for example Zaiss et al. (Journal of Virology 7209-7219 (2002)) and Schambach, Baum et al. (Molecular Therapy 2007, 15(6): 1167-73). This is also important in that the U3 region aside from promoter elements and enhancer elements also contains polyadenylation enhancers and therefore any deletions have to be validated anew for each virus family with regard to residual enhancer activity and efficiency of the polyadenylation. 
     For gammaretroviral vectors Kraunus et al. (Gene Therapy 568-1578 (2004)) propose to delete the complete region of the enhancer element and the promoter of the 3′ U3 region and to leave only the first 22 bp upstream of the enhancer and the last downstream 14 bp. With ASLV and rous sarkoma virus (RSV) the LTR region is complicated by a very short R region such that the polyA signal (AATAAA) comes to lie in the U3 region. This is very unusual for retroviruses (otherwise located in the R region) and makes the construction of a SIN vector more difficult. 
     Aubert et al. (The J. of Cell Biology 113, 497-506 (1991) and Flamant et al. (Journal of General Virology 74, 39-46 (1993)) describe deletions of the U3 region of ALV from −149 to −15 relative to the R region, and from −149 to −15, and −98 to −59, respectively, again relative to the R region. In all these U3 regions remaining enhancer elements are still contained (Cullen et al., MCB 1985, 438-47) which is problematic for possible clinical applications. This is because these enhancer elements might dysregulate adjacent genes (e.g. oncogenes) by insertional mutagenesis. 
     OBJECT OF THE INVENTION 
     It is therefore the object of the invention to provide a clinically suitable split-packaging system having a viral vector and helper plasmids for the production in mammalian cells (e.g. human cells, for example HEK293T) by which the viral vector can be produced packaged as a viral particle, in which the risk of the recombination to a replication-competent retrovirus (RCR) is minimized, and also the risk is minimized that the insertion of the viral vector leads to the activation of adjacent genes of the target cell (insertional mutagenesis). Preferably, the viral vector and the system having helper plasmids, respectively, shall furthermore allow the generation of viral particles at a high titre, wherein the viral vector shall be able to contain an expression cassette from which a transgene and gene regulatory sequences (e.g. shRNA, miRNA etc.) are efficiently transcribed and/or translated. Preferably, these vectors are pseudotyped with clinically suitable envelope proteins (e.g. RD114/TR, MLV amphotropic env, glycoprotein of the vesicular stomatitis virus (VSVg)). All these criteria are aspired in combination, since they are essential for possible clinical applications. 
     GENERAL DESCRIPTION OF THE INVENTION 
     The invention achieves this object by the features of the claims, and especially by a vector having the aspired properties. A viral RNA can be transcribed from a plasmid according to the claims providing a viral vector on the basis of ASLV, the 3′ U3 region of which produces or is, respectively, a self-inactivating 3′ LTR (SIN LTR), as well as providing the viral particle containing a nucleic acid sequence encoding the viral vector, especially RNA. Furthermore, the invention provides a system having a first helper plasmid encoding in an expression cassette the coding sequence for the gag-pro-pol fusion protein of ASLV, and having a second helper plasmid encoding in an expression cassette an envelope protein (env), e.g. env of ASLV, RD114/TR, MLV Amphotrop, VSVg or MLV Ecotrop. 
     Upon presence of the first and of the second helper plasmids in a eukaryotic cell, in the following also denoted as packaging cell, viral particles are produced at high titre the viral RNA of which itself is self-inactivating (SIN) due to the deletions in the 5′ U3 region originating in a target cell from the 3′ U3 region during the reverse transcription to the viral DNA, but allow a high transcription rate and efficient translation of the transgene which is arranged in an expression cassette in 5′ before the 3′ SIN LTR. In this configuration the activation of adjacent genomic sequences by the insertion of the viral vector is significantly reduced. The reduction of the activation of adjacent sequences has been shown for γ-retroviral vectors by Zychlinski et al. (Mol. Ther. 16: 718-725 (2008)). 
     Mutations which are known for viral SIN vectors on the basis of other retroviruses, for example lentiviruses, which combine the properties of the high titre in packaging cells on the one hand, no promoter activity in the LTR, as well as an efficient transcription of a transgene from a vector-internal expression cassette without through-transcription into genomic sequences at the place of insertion, cannot be transferred to the construction of SIN LTR vectors on the basis of ASLV. This is because the arrangement of functional elements in the individual viruses and their interaction with cellular host factors are not identical to each other and the effect of the change of sequences to the titre, to the prevention or the activation of adjacent genomic sequences at the place of insertion, and to a transcription of the transgene from an internal expression cassette cannot be predicted. 
     The viral vector according to the invention in the place of the wild-type LTR contains a 3′ LTR the U3 region of which is deleted over a large section such that the transcriptional activity of the promoter and enhancer regions of the U3 region is essentially, preferably completely, turned off and eliminated, respectively. The elimination of the promoter and enhancer regions of the U3 region, e.g. by deletion or replacement of nucleotides of this region, is determined by means of an activity test when the promoter activity of the deleted or substituted U3 region is on the level of the background activity of a promoterless reporter gene, as e.g. for a completely deleted U3 region for a reporter gene subsequently arranged functionally in 3′. In particular, according to the invention the U3 region of ASLV only has nucleotides 1-27 and nucleotides 187-229, preferably only nucleotides 1-27 and 207-229 of the 229 nucleotides of the wild-type U3 region (SEQ ID NO: 1). This altered 3′ U3 region of the viral vector according to the invention, in which at the wild-type U3 region nucleotides 28-186 are deleted, and preferably in addition nucleotides 187-202 are deleted, after reverse transcription and integration into the genome of a target cell essentially has no promoter activity and/or enhancer activity. The packageable viral RNA is driven by a strong promoter arranged in 5′ which is lost in the course of the reverse transcription and is not contained in the viral RNA produced by transcription, respectively. This allows the generation of a transcript which in combination with a first and a second helper plasmid is suitable for the generation of viral particles having a high titre which contain the viral RNA. 
     The deletion of promoter regions and of enhancer regions optionally can be substituted by an insertion with a nucleotide sequence having no promoter activity or enhancer activity. 
     The 3′ SIN LTR of the vector according to the invention can consist of the aforementioned sections of the deleted wild-type U3 region, a wild-type R region and a wild-type U5 region. The U3 region having deletions according to the invention optionally can contain additional nucleotide sequences without promoter activity between the remaining sections of the wild-type U3 region, which for example are selected from the group comprising miRNA, shRNA, polyadenylation enhancers (USE), recombinase recognition sequences (e.g. LoxP, Rox, FRT) and insulator elements (e.g. cHS4). 
     For insertion of nucleotide sequences having no promoter activity into the U3 region according to the invention this region preferably has an unique cutting site for a restrictase, especially preferred in that the nucleotides 187-190 in the numbering of the wild-type U3 region through mutation produce the base sequence CGTA, whereby a secondary cutting site for SnaBI having the recognition sequence TACGTA is generated. 
     As an alternative to the deletion of the nucleotides 187-202, also the nucleotides 200-206 (TATTTAA) can be mutated, for example into the sequence TGTCTAA. It is assumed that the nucleotides 200-206 form the TATA box of the wild-type U3 region, so that a mutation which changes this TATA box reduces or abolishes this portion of the promoter activity of the U3 region. 
     The deleted U3 region according to the invention further has an integrase attachment site (integrase attachment), especially in nucleotides 1-25, as well as a polyA signal, e.g. at nucleotides 223-228, each in the numbering of the nucleotides of the wild-type U3 region. It is assumed that the maintenance of the integrase attachment site and of the polyA signal is important for the viral infection. Surprisingly, the vector according to the invention on the one hand allows a production of viral particles at a high titre in a packaging cell or packaging cell line, and on the other hand allows an efficient transcription of a transgene from an expression cassette from an internal strong promoter following reverse transcription and integration into the genome of a target cell, without a through-transcription over the 3′ SIN LTR occurring into adjacent regions of the integration site or activation of adjacent genomic sequences. 
     Viral particles produced in packaging cells which contained viral RNA having a 3′ SIN LTR according to the invention or a 3′ U3 region according to the invention, without concentration had a titre of approximately 1×10 6  infectious particles/mL. Therein the titre was influenced by the gene encoding the gag-pro-pol fusion protein and the envelope protein, respectively, of the first and the second helper plasmid, respectively, and was not significantly influenced by the nature or extent of the deletion in the U3 region. 
     Surprisingly, the expression of a transgene from an expression cassette in viral vectors according to the invention having a SIN deletion was higher by approximately a factor of 3 than in a comparative vector, in which the 3′ LTR had a U3 region of wild-type sequence. According to the invention it is further preferred that the coding sequences of the first and/or of the second helper plasmid are codon-optimized such that the viral codons are substituted by codons of the target cell, especially are substituted by human codons, e.g. in methods for the production of viral particles and of viral vectors according to the invention which e.g. find a use as medicament or in the production of pharmaceutical compositions. A preferred helper plasmid according to the invention contains a sequence encoding the gag-pro-pol fusion protein in which viral codons are adapted to the human codon usage. 
     In the use as a medicament the viral particle containing the viral vector can be prepared for administration e.g. to a patient/human or to a non-human animal. Alternatively, the viral particle and the viral vector, respectively, can be contacted with cells originating from such a patient, i.e. for in vitro transduction of cells which subsequently, optionally with the step of cultivation and/or selection, are prepared and provided, respectively, as transduced cells for transfer into a patient, wherein preferably the patient is the same from which the cells originate. 
     According to the invention in the codon-optimized coding sequence for the gag-pro-pol fusion protein the initial shift of the reading frame by −1 and the transition from pro to pol from the wild-type ASLV has been maintained. 
     It has been found that the adaptation of the codon usage of ASLV to the codon usage of the target cell, especially to the human codon usage, for the coding regions of the helper plasmids results in a significant increase of the titre of viral particles, wherein e.g. the adaptation of the codon usage of the fusion protein gag-pro-pol to the human codon usage by itself leads to a titre of viral particles in human packaging cells which is higher by a factor of about 50. 
     The adaptation of the codon usage to the human codon usage preferably occurs by exploiting the optimal non-limited tRNA pools of homo sapiens: e.g. the glycin codon GGT was preferably adapted to GGC. Furthermore, negative RNA instability motives as well as cryptic polyadenylation sites as well as RNA secondary structures were removed, such that a more stable mRNA having better translatability in human cells was generated. Furthermore, in the coding sequence the natural splice donor site, the splice acceptor site and further cryptic splice signals were deactivated. Accordingly, a preferred helper plasmid has an inactive splice donor site in the gag-pol gene, e.g. corresponding to nucleotides 1501 to 1502 in SEQ ID NO: 14. Preferably, gag is encoded by nucleotides 1475 to 3436 of SEQ ID NO: 14 and pol is encoded by nucleotides 3738 to 6291 of SEQ ID NO: 14, since in these sections cryptic RNA instability motives and polyadenylation sites as well as RNA secondary structures have essentially been removed, such that a high titre of viral particles having a high transduction efficiency can be produced. 
     Finally, it is preferred in the invention that the viral vector, preferably in addition the first helper plasmid and further preferred in addition the second helper plasmid, have no sequence homologies to the target cell, and further preferred in particular have no sequence overlap, e.g. no sequence sections of more than 5 nucleotides homologous to each other, preferably no sequence sections of more than 10 to 20 nucleotides homologous to each other. In this way the probability of a recombination in packaging cells and/or in the target cells is minimized and thereby the probability of generation of RCR is minimized, too. 
     Accordingly, the invention provides a viral RNA which from 5′ to 3′ has or consists of a 5′ R region and a 5′ U5 region, a primer binding site (PBS), a packaging signal (Ψ) which in contrast to the state of the art of gammaretroviral or lentiviral vectors suffices without a retroviral splice donor, an expression cassette for a transgene or comprising a transgene, respectively, and a shortened 3′ U3 region (marked as Δ or d) according to the invention, a 3′ R region and an appending polyA section (polyA tail), as well as a vector for transcription of the viral RNA which in addition to a nucleic acid sequence encoding the viral RNA in 5′ to its 5′ R region has a eukaryotic or viral promoter, respectively, and in which vector the nucleic acid sequence encoding the viral RNA is present in the form of DNA which in the 3′ SIN LTR has a polyA section. 
     Furthermore, the invention provides the use of the viral RNA and of a viral particle containing the viral RNA as a medicament and for the production of a pharmaceutical composition for use for the transduction of target cells, e.g. of somatic cells of a human, as well as pharmaceutical compositions comprising the viral RNA or viral particles containing the viral RNA, and the use of such pharmaceutical compositions as a medicament for the transfer of the transgene into cells, especially for gene therapy. 
     Furthermore, the invention relates to cells having an integrated or non-integrated nucleic acid sequence which are produced by contacting with viral particles according to the invention and which contain a U3 region according to the invention, e.g. a DNA section resulting from reverse transcription from a viral RNA according to the invention. These can be extracorporeal animal cells or intracorporeal animal cells, with the exception of human gametes. 
     The expression cassette has a promoter and at least one transcribable and/or translatable sequence of a transgene, which can be a gene homologous or heterologous to the target cell, and preferably upstream of the transcribable sequence of the transgene a PRE or other posttranscriptional regulatory nucleic acid sections or nucleic acid modules. The transcribable sequence can also have internal ribosome entry sites (IRES) in combination with protein encoding sequences arranged downstream to these, as well as other functional nucleic acid sections or enzyme encoding nucleic acid sections (e.g. bidirectional promoters, coding sequences for 2A protease cutting sites or 2A protease genes). Further optionally, an insulator sequence can be inserted into the deleted 3′ U3 region. e.g. into the restriction site contained therein, which weakens an interaction of the transgene with adjacent genomic sequences, or a recombinase recognition sequence can be inserted, e.g. a recognition sequence from the group comprising rox, loxP, FRT. After reverse transcription and copying into the 5′ LTR has occurred, the recombinase recognition sequence can be activated by addition and expression of recombinase in target cells, respectively, and e.g. a nucleic acid section in the integrated DNA (e.g. a portion of the expression cassette) arranged between recombinase recognition sequences can be turned around or removed. Other sequences which can be inserted into the 3′ U3 region for example comprise micro-RNA target sequences, small hairpin RNAs or sequence sections which make the detection of the insertion by PCR easier. 
     The deleted U3 region contained in the 3′ SIN LTR according to the invention in spite of the deletion of the enhancer and promoter elements from the wild-type U3 region allows the generation of viral particles having titres which are achieved comparatively to the titres for vectors having wild-type LTR in packaging cells, so that the drastic reductions of the viral titre frequently observed upon deletions of U3 regions are avoided. 
     The transcription of transgenes which are arranged in the expression cassette is controlled by the promoter of the expression cassette which is also termed an internal promoter. Presently it is assumed that the deleted U3 regions according to the invention have a polyA signal which avoids a through-transcription beyond the vector sequence and uses the correct polyA signal. This assumption is supported by the 3-fold improved expression of a transgene of the expression cassette of a vector according to the invention in comparison to the expression of the transgene from a vector having a 3′ LTR of the wild-type. 
     Particularly preferred, the nucleic acid sequence of the viral vector according to the invention has a complete deletion of the sequences encoding viral structural proteins including the splice donor site (SD), e.g. a complete deletion of the sequences gag, env, pol and src. It has been found that with ASLV vectors having a deletion of the sequences encoding the viral structural proteins also the splice donor site is deleted. Surprisingly, it has been found that such a vector on the basis of ASLV allows the packaging of the viral RNA in packaging cells by means of the split-packaging system. This is unexpected insofar as in known retroviral vectors portions of the coding viral sequences are required for the packaging, since in the region of the sequences encoding the viral structural proteins signals are contained for the RNA export, for the interaction with the viral nucleocapsid and for the reverse transcription, as e.g. in HIV. Therefore, the vector according to the invention can contain the packaging signal (Ψ) which is comprised in SEQ ID No: 5 by the nucleotides 1042-1292 without sections of sequences encoding viral structural proteins. 
     According to the invention the preferred embodiment in which the nucleic acid sequence of the viral vector has no viral splice donor is an important difference to known lentiviral or γ-retroviral vectors which have the natural viral splice donor upstream of the primer binding site. These known vectors require the splice donor for the generation of high titres of viral particles. Surprisingly, it has been found that the vector according to the invention results in high titres in packaging cells even without its natural or another splice donor. Since the natural splice donor of ASLV is located in the gag gene, its complete deletion consequently leads to the removal of the splice donor. Further preferred according to the invention also the viral splice acceptor is not contained in the vector. The splice acceptor of ASLV is located in the pol gene, so that its complete deletion also leads to the removal of the splice acceptor. 
     For the vector according to the invention the deletion of splice donor and splice acceptor leads to the optimisation of the production of viral particles in packaging cells and in addition gives a better biological safety to the vector, since the recombination to replication-competent retroviruses is made more difficult. Also a possible interference with cellular splice signals is less probable in intragenic insertions of the vector. 
     Particularly preferred, the vector in the 5′ region has an R region, a U5 region and the packaging signal in a nucleic acid sequence which does not encode viral structural proteins and to which in 3′ the expression cassette of the transgene adjoins e.g. directly, especially following the leader region. It has been found that the viral vector at its 5′ terminus can have an R region, a U5 region and a packaging signal which is comprised or consists of nucleotides No. 1042 to 1292 of SEQ ID NO: 5, wherein preferably in 3′ adjacent to this sequence the expression cassette having the transgene is arranged. 
     For the purposes of the invention the arrangements of genetic elements are given in 5′ to 3′, as long as not described differently; nucleotide sequences are given from 5′ to 3′. 
    
    
     
       DETAILED DESCRIPTION OF THE INVENTION 
       The invention is now described in greater detail by way of Examples with reference to the Figures in which 
         FIG. 1  shows the schematic structure of the ASLV wild-type, 
         FIG. 2  shows the schematic structure of a SIN vector according to the invention and of a plasmid containing the vector, 
         FIG. 3  shows the schematic structure of a first helper plasmid, 
         FIG. 4  shows the schematic structure of a second helper plasmid, 
         FIG. 5  shows a comparison of the wild-type U3 region (U3, SEQ ID NO: 1) to the deleted U3 region (dU3, SEQ ID NO: 2) according to the invention, to the deleted U3 region according to the invention without TATA box (dU3 noTATA, SEQ ID NO: 4) and to the deleted U3 region according to the invention having a mutated TATA box (dU3 TATAmut, SEQ ID NO: 3), 
         FIG. 6  shows a plasmid map of a vector (SEQ ID NO: 5) according to the invention in which the expression cassette encodes EGFP (SEQ ID NO: 6) as an example for a transgene, 
         FIG. 7  shows a plasmid map comprising the vector (SEQ ID NO: 9) according to the invention in which the TATA box of the U3 region is mutated, 
         FIG. 8  shows a plasmid map comprising the vector (SEQ ID NO: 7) according to the invention in the U3 region of which additionally the TATA box is deleted, 
         FIG. 9  shows a plasmid map for a first helper plasmid (SEQ ID NO: 11) for translation of the gag-pol fusion protein, 
         FIG. 10  shows a plasmid map for a first helper plasmid (SEQ ID NO: 14) in which the sequence encoding the gag-pol fusion protein is codon-optimized for a human packaging cell and/or target cell, 
         FIG. 11  graphically shows the level of the expression of a transgene (EGFP) from the vector according to the invention in comparison to the expression from a viral vector having a wild-type 3′ LTR, 
         FIG. 12  graphically shows the level of expression of a transgene (EGFP) from SIN vectors according to the invention, 
         FIG. 13  shows a depiction of the integrated structure of a vector according to the invention in comparison to the structure of a lentiviral and γ-retroviral SIN vector, respectively, 
         FIG. 14  schematically shows DNA nucleic acid constructs for the transcription of viral RNA having expression cassettes which functionally contains the nucleic acid sequence to be tested for promoter activity and enhancer activity in 5′ to a reporter gene, and 
         FIG. 15  shows the level of expression of the reporter gene in cells which are transfected with viral RNA of  FIG. 14  and measures the promoter activity/enhancer activity. 
     
    
    
     In the description of the SIN U3 according to the invention the numbering of the nucleotides is carried out with reference to the nucleotide sequence of the wild-type U3 of ASLV and correspondingly, deletions and sections of the SIN U3 region, respectively, are denoted according to the numbering of the nucleotides of the wild-type U3. 
       FIG. 1  shows the schematic structure of the ASLV of the wild-type in which the LTRs contain an U3 region, an R region and a U5 region adjacent to each other. The LTRs are identical each time, since in the viral replication the U3 region of the 3′ LTR is copied to the 5′ terminus and the regions R and U5 of 5′ LTR are copied into the 3′ LTR. The transcription start +1 defines the 5′ terminus of the 5′ R region. 
       FIG. 2  shows the vector according to the invention at plasmid level at the 5′ terminus of which a promoter is arranged ahead of the R region in order to enable the transcription starting from the 5′ R region. This 5′ promoter of the 5′ LTR due to the transcription start at +1 as first nucleotide of the 5′ R region is not contained in the viral RNA; the viral RNA which is produced consists of the regions between the nucleotide +1 of the 5′ R region and the 3′ terminus of the 3′ R region plus polyA tail. Correspondingly, viral particles contain a viral RNA which is generated from that section of the vector which comprises the 5′ R region (including nucleotide +1) up to the 3′ terminus of the 3′ R region with an appending polyA tail. The promoter of the viral vector upstream of the 5′ R region shown in  FIG. 2  which preferably consists of an SV40 enhancer and of a U3 region of the wild-type of the RSV (Rous Sarcoma Virus) does not become a component of the viral RNA, but controls the efficient transcription of the viral RNA of a DNA sequence corresponding to  FIG. 2 . In the present case the vector is shown at the plasmid level as a DNA sequence encoding the viral RNA which can be a component of a plasmid from which the viral RNA is transcribed. 
     As indicated in  FIG. 2 , the viral vector between the LTRs and correspondingly after the leader region contains an expression cassette which comprises at least one internal promoter, for example the promoter of SFFV and EFS (elongation factor, 1α) having a nucleotide sequence encoding a transgene (gene), preferably having a PRE arranged in 3′. 
     The PRE which preferably is WPRE of the expression cassette preferably has an X-ORF deletion and/or ATG mutations. 
     The vector according to the invention and the viral RNA according to the invention at least in the 3′ LTR have a U3 region having deletions which deactivate any promoter activity of the LTR. In 5′ to the 3′ SIN LTR and/or within the 3′ U3 region the viral vector according to the invention can contain homologous and/or heterologous genetic elements, for example sequences which comprise miRNA, shRNA, polyadenylation enhancers, recombinase recognition sequences, USE and/or insulators. 
     Between the elements of the 5′ LTR and the 3′ LTR, preferably between the regions of the 5′ LTR and the expression cassette, the vector according to the invention preferably has a primer binding site and a packaging signal (Ψ). 
     In contrast to replication competent retroviruses the viral vector according to the invention has no sequences coding for gag-pol and/or envelope proteins. 
     The viral proteins which are necessary for the generation of a viral particle of the viral RNA are encoded by separate helper plasmids, preferably by a first helper plasmid which has a coding sequence for the gag-pro-pol fusion protein in an expression cassette between a promoter and a polyadenylation sequence, as depicted in  FIG. 3 , and by a second helper plasmid containing a sequence encoding the envelope protein (env) in an expression cassette, for example as depicted in  FIG. 4  between a promoter of CMV (cytomegalovirus) and a polyadenylation sequence. For pseudotypification the sequence encoding the envelope protein can consist of a sequence encoding the envelope protein of MLV Ecotrop/Amphotrop, VSVg, RD 114, GALV or chimaera of these, e.g. RD114/TR. 
     The first helper plasmid in its coding sequence preferably comprises the reading frame shift of −1 between the 3′ terminus of the sequence encoding gag-pro and the 5′ terminus of the sequence encoding pol, which is shown in  FIG. 3  for gag-pro and pol, respectively, by the staggered boxes. 
     Particularly preferred, the coding sequences of the helper plasmids are adapted to the codon usage of the target cell, i.e. codon-optimized, since it has&#39;been found for the invention that this codon-optimization for the gag-pro-pol fusion protein, preferably also for the envelope protein env, results in an increase of the titre in helper cells approximately by the factor of 50. Therefore, the invention enables a production method in titres and amounts of viral particles, respectively, as they are required for clinical applications, by production of viral particles containing the viral RNA according to the invention, by a first helper plasmid and a second helper plasmid, e.g. by culturing of a packaging cell. 
       FIG. 5  shows an overview of the preferred sequences of the 3′ U3 region of the viral vector according to the invention in comparison to the sequence of the wild-type U3 (SEQ ID NO: 1) of ASLV. The U3 regions according to the invention preferably have a deletion of the nucleotides 28-186 of the wild-type U3. The embodiment SEQ ID NO: 2, denoted as Delta (d) U3, preferably consists of the nucleotides 1-27 and of the nucleotides 187-229 of the wild-type U3, wherein the nucleotides 187 and 189 are further mutated to C and T, respectively, such that a restriction site for SnaBI (TACGTA) results there. This deleted U3 region, also denoted as SIN U3, still has the initial TATA box which was identified as nucleotides 200-206 of the wild-type U3. 
     In a preferred embodiment the TATA box of the SIN U3 is mutated, too, such that the function of a TATA box is annulled. A preferred embodiment of a mutated TATA box is shown in SEQ ID NO:3 (TdU3 TATAmut) in which the nucleotides 201-203 are mutated, such that the function of the TATA box is destroyed. 
     In a preferred embodiment the deletion with respect to the wild-type U3 region comprises the nucleotides 28-205 (dU3 noTATA; SEQ ID NO: 4), wherein the nucleotides 203-204 are mutated in order to avoid the generation of a TATA box by the adjoining sections and/or in order to generate a cutting site at the adjoining nucleotides 26-27 and 203-206, for example by mutation of the nucleotides 203-205 to CGT in order to generate a singular cutting site for the restriction enzyme SnaBI (TACGTA). 
     Viral vectors according to the invention therefore also comprise variants in which in the region of the deletion of nucleotides 28-222, preferably in the region of the nucleotides 28-190 and 28-206 in SEQ ID NO: 1, respectively, heterologous or homologous, e.g. genetic elements heterologous or homologous with respect to the target cell, can be inserted, preferably by means of a restriction site in the aforementioned sections. 
     Example 1 
     Production of Viral Particles Having an ASLV SIN Vector According to the Invention 
     As examples for vectors according to the invention coding for a viral RNA according to the invention plasmids were constructed which under the control of a promoter of an enhancer element of SV40 (SV40 enhancer) and the RSV promoter (RSV) directly adjacent to the promoter had the R region, the U5 region, an expression cassette for a transgene and a 3′ SIN LTR from a SIN U3 region according to the invention, an R region and a U5 region. The expression cassette comprised the U3 promoter of the spleen focus-forming virus (SFFV), a sequence coding for EGFP as an example for a transgene, and a WPRE (PRE). 
     For the vector shown in  FIG. 6 , the SIN U3 region corresponded to the SEQ ID NO: 2, alternatively to the SEQ ID No: 3 for the vector shown in  FIG. 7 , and corresponded to the SEQ ID NO: 4 for the vector shown in  FIG. 8 , respectively. 
     The sequence of the vector of  FIG. 6  is contained as SEQ ID NO: 5. Correspondingly, a vector according to the invention and a plasmid encoding an RNA according to the invention, respectively, contains a 3′ SIN LTR having a sequence corresponding to nucleotides 3282-3452 of SEQ ID NO: 5. The viral RNA transcribed therefrom contains the 3′ U3 region according to the invention contained therein. 
     The sequence of the vector of  FIG. 7  is contained as SEQ ID NO: 9. Correspondingly, a vector according to the invention from which a viral RNA according to the invention is transcribed contains a 3′ SIN LTR having a sequence corresponding to the nucleotides 3282-3452 of SEQ ID NO: 9. The viral RNA contains the 3′ U3 region and R region according to the invention at the 3′ SIN LTR, but not the U5 region. 
     The sequence of the vector of  FIG. 8  is contained as SEQ ID NO: 7. Correspondingly, a vector according to the invention from which a viral RNA according to the invention is transcribed contains a 3′ SIN LTR having a sequence corresponding to the nucleotides 3282-3436 of SEQ ID NO: 7. 
     Further preferred, a viral vector and a viral RNA, respectively, in the form in which the nucleotide sequence for the viral RNA is contained on a plasmid, contain a wild-type R region and a wild-type U5 region as the 5′ LTR and directly adjacent thereto in 5′ a eukaryotic or viral promoter, preferably the enhancer sequence from SV40 in combination with the promoter from RSV, corresponding to nucleotides 452-921 of SEQ ID NO: 9. 
     The sequence of SEQ ID NO: 5 coding for eGFP is given as amino acid sequence SEQ ID NO: 6, eGFP of SEQ ID NO: 7 as SEQ ID NO: 8, and eGFP of SEQ ID NO: 9 as SEQ ID NO: 10. 
     For generation of viral particles the plasmids according to  FIGS. 6 to 8  were introduced into HEK293T cells which also contained a first helper plasmid for expression of the gag-pol fusion protein of ASLV by means of transient transfection or stable integration. A schematic plasmid map is shown in  FIG. 9 , the sequence is contained as SEQ ID NO: 11 and contains the gag-pol fusion protein at nucleotides 1475-6291 as coding sequence. The amino acid sequence of gag encoded by SEQ ID NO: 11 corresponds to SEQ ID NO: 12, the one of poi encoded by SEQ ID NO: 11 corresponds to SEQ ID NO: 13. 
     Particularly preferred, the first helper plasmid was provided with a coding sequence for the gag-pol fusion protein the codons of which were adapted to the codon usage of the packaging cell, especially of mammalian cells, as e.g. human cells. Such a plasmid is schematically shown in  FIG. 10 ; the nucleic acid sequence is contained as SEQ ID NO: 14. Correspondingly, a preferred first helper plasmid and a human packaging cell, respectively, in integrated form contain the coding sequence for the codon-optimized gag protein corresponding to nucleotides 1475-3436 of SEQ ID NO: 14, the amino acid sequence of which corresponds to SEQ ID NO: 15, and/or the coding sequence for the codon-optimized pol protein corresponding to nucleotides 3738-6291 of SEQ ID NO: 14, the amino acid sequence of which corresponds to SEQ ID NO: 16, preferably the coding sequence for the codon-optimized gag-pol fusion protein corresponding to nucleotides 1475-6291 of SEQ ID NO: 14. 
     Under usual cultivation conditions of the packaging cell the same titres were obtained each time for the viral particles according to the invention having one of the SIN U3 regions shown of the vectors of SEQ ID NO: 7, 9 or 11, as for a comparative construct (not shown) in which the 3′ U3 region corresponded to the wild-type of ASLV, namely approximately 1×10 6 /mL. 
     It is therefore preferred for the method for production of viral particles according to the invention that the packaging cells are cultured human cells having a nucleic acid sequence encoding the viral structural genes, e.g. gag-pol, having human codon usage, wherein these structural genes preferably have no active splice donor site and/or splice acceptor site, in order to avoid a splicing within the coding sequences and to thereby achieve a high expression in the packaging cells. 
     For control of the transfection of target cells with viral RNA human HT1080 fibroblasts were infected. Subsequent to the cultivation after the transfection the expression of the transgene (EGFP) was determined by FACS. In the comparison to viral particles the 3′ LTR of which was of wild-type ASLV, an expression of the transgene higher by the factor of 3 could be shown for the viral vectors having 3′ SIN LTR according to the invention. 
       FIG. 11  shows the expression of the exemplary transgene EGFP by an expression cassette according to  FIG. 7  in target cells which were transduced with viral vector. Viral particles were obtained from the supernatant of packaging cells (pseudotyped with VSVg) which contained a plasmid encoding viral RNA according to SEQ ID NO: 5 according to the invention (ASLV dU3, bright right bars), and a viral RNA having a 3′ U3 of the wild-type (ASLV U3, dark left bars; wild-type U3 see SEQ ID NO:1). For transduction viral particles were titrated onto HT1080E cells, cultivated for 6 days and analysed flow-cytometrically subsequently. In Experiment 1 the gag-pol fusion protein was of the wild-type (nucleotides No. 1475 to 6291 in SEQ ID NO: 11), in Experiment 2 it was codon-optimized (nucleotides No. 1475 to 6291 in SEQ ID NO: 14). The result was that the viral particle according to the invention having a 3′ U3 region according to the invention in comparison to the viral particle having a U3 region of the wild-type resulted in a 3-fold higher expression of the transgene in target cells. This applies both to the use of the wild-type gag/pol (Experiment 1) and to the codon-optimized gag-pol helper plasmid (Experiment 2). In both cases an approximately 3-fold increase of the expression of the transgene in target cells results for viral particles according to the invention (dU3) in comparison to the wild-type comparative particles (U3). 
     
       
         
               
               
             
               
               
               
             
           
               
                   
                   
               
               
                   
                 average fluorescence intensity 
               
             
          
           
               
                 experiment 
                 ASLV U3 
                 ASLV dU3 
               
               
                   
               
               
                 1 (wild-type gag-pol) 
                 503.8 +/− 15.1 
                 1748.2 +/− 112.6 
               
               
                 2 (codon-optimized gag-pol) 
                 791.3 +/− 48.7 
                 2275.6 +/− 120.6 
               
               
                   
               
             
          
         
       
     
       FIG. 12  shows the values of the expression of the exemplary transgene EGFP by an expression cassette in target cells transduced with viral vector which contained viral RNA transcribed from a plasmid according to SEQ ID NO: 5 (SIN, according to the invention), SEQ ID NO: 9 (TATAmut), and SEQ ID NO: 7 (noTATA), respectively. As described above, viral particles were generated in packaging cells which translated codon-optimized gag-pol fusion protein and VSVg envelope protein. HT1080 cells were transduced with viral particles, cultivated for 6 days, and then analysed by FACS. The values shown make it clear that the 3′ U3 region according to the invention in all viral RNAs and viral particles according to the invention, respectively, results in a high expression efficiency of the transgene in target cells. The results are also presented in the following table: 
     
       
         
               
               
             
               
               
               
             
           
               
                   
                   
               
               
                   
                 average fluorescence intensity 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 ASLV dU3 
                 3616.9 +/− 15.8  
               
               
                   
                 ASLV dU3 TATAmut 
                 3020.4 +/− 227.8 
               
               
                   
                 ASLV dU3 noTATA 
                 3463.2 +/− 252.8 
               
               
                   
                   
               
             
          
         
       
     
     The preferred embodiment of the vector according to the invention, e.g. its viral RNA, optionally contained in viral particles, has no coding viral sequences, e.g. no sequences completely or partially comprising one of the structural genes gag, env, src or pol. Therefore it is preferred that the viral RNA of the vector and the transcribed nucleic acid sequence of a plasmid encoding the viral RNA of the vector, respectively, the 5′ R region, the 5′ U5 region and the region comprising the packaging signal and extending adjacent to the expression cassette of the transgene consist of these regions and e.g. do not contain a splice donor site. A preferred sequence for the 5′ R region, the 5′ U5 region and the region comprising the packaging signal and extending up to adjacent to the expression cassette of the transgene comprises the nucleotides No. 922 to 1292 of SEQ ID NO:5 or consists of this nucleotide sequence. This nucleotide sequence optionally can be shortened in 3′, e.g. by 140 nt, preferably by 10 to 56 nt. 
     In this embodiment in which the region of the viral RNA at its 5′ terminus and therefore in 5′ directly adjacent to the expression cassette of the transgene, consists of non-translated sequences, especially of an R region, a U5 region and the packaging signal, therefore no sections of gag, env, src or pol are contained. 
     This embodiment is schematically shown in  FIG. 13 , making it clear that this 5′ section of the vector which is also denoted as leader, is significantly shorter than the 5′ region of lentiviral or γ-retroviral SIN vectors. Whereas in lentiviral and many versions of γ-retroviral SIN vectors the packaging signal Ψ overlaps with viral structural genes, in the alpharetroviral vector according to the invention, surprisingly, the packaging signal can be contained without coding viral sequence sections. Therefore, the vector according to the invention (alpharetroviral SIN vector) is denoted as a gag-free leader. Furthermore, in the vector according to the invention the viral splice donor site can be removed and made functionless by mutation, respectively. E.g. in lentiviral or γ-retroviral vectors this is possible only under reduction of the titre due to overlap with packaging functions. Furthermore, the complete removal of coding viral sequence sections allows the shortness of that section of the vector sequence which is arranged in 5′ prior to the expression cassette of the transgene, e.g. of 371 nt (nucleotides, bp). Therefore, in comparison to lentiviral and γ-retroviral vectors particularly much room is generated for the intake of transgene sequences. The expression cassette in these examples consists of the strong internal promoter of SFFV, the coding sequence for green fluorescent protein (EGFP) as a reporter gene and the WPRE each time. 
     Example 2 
     Long Term Expression of the Transgene in Eukaryotic Target Cells 
     As an example for the use of the viral vector as a medicament, e.g. for gene therapy, blood stem cells were transduced with viral particles according to the invention. Isolated bone marrow cells from mice (strain C57BL6) were transduced with viral particles according to the invention, and for comparison with lentiviral particles. As a transgene EGFP was contained each time in the same expression cassette under the control of the SFFV promoter. Under conditions of same efficiency the cells were transfected by the lentiviral vector to 39% and therein by the factor of 1.6 more than by the vector according to the invention (24%). This can be attributed to the fact that lentiviral particles can also transduce resting, divisionally inactive cells, while the α-retroviral particles according to the invention preferably can transduce cells during the division. 
     The transduced blood stem cells were transplanted into 8 lethally irradiated mice of the same strain. Transduced cells were identified from peripheral blood by FACS. After 31 weeks the proportion of transduced EGFP-positive leukocytes for lentiviral vectors was increased at 32.9+/−8.9% to approximately double the value of the vectors according to the invention (16.1+/−12%). 
     From both groups bone marrow was taken from the animals having the highest titres of EGFP-positive cells and transplanted into 5 lethally irradiated animals of a second group each. 6 weeks after this transplantation the proportion of EGFP-positive leukocytes for the lentivirally transduced stem cells at 40.6+/−5.9 still was approximately 30% above the Proportion of transduced stem cells according to the invention (32.6+/−8.1%). This shows that the velocity of the epigenetic shutdown of the expression of the transgene for the vectors according to the invention is not essentially higher than for lentiviral vectors. 
     Example 3 
     Demonstration of Lower Risk by Insertional Mutagenesis of the Vectors 
     In an in vitro test developed by the inventors (Mol. Ther. 1919-1928 (2009)) the risk of oncogene activation in the transduction and transfection by the viral vector, respectively, was examined. An oncogene activation, e.g. by insertion of viral sequences into the genomic neighbourhood, therein leads to the transformation of primary haematopoietic cells. In short it is investigated in this test in which frequency and to which degree of distinctness the insertional activation of transformation supporting cellular proto-oncogenes like EviI occurs. In this test primary haematopoietic cells of the untreated mouse, e.g. strain C57BL6, are contacted with viral particles. After this transduction the transformation frequency is determined by cultivation under conditions which cause the myeloid differentiation, wherein the renewed seeding is carried out from such cell dilutions in which non-transformed cells do not proliferate due to the inhibition of the remaining capability of division caused by the dilution, but only transformed cells proliferate. In transformed cells e.g. the expression of proto-oncogenes is up-regulated by insertional mutagenesis. This test therefore quantifies the transformation frequency in relation to transduced cells. The viability and stability of the transformants, respectively, therefore can be determined as the result of the cultivation of transformed cells after renewed seeding and replating, respectively. 
     It has been found therein that γ-retroviral vectors and lentiviral vectors lead to a transformation frequency of approximately 1×10 −5  and 5×10 −6 , respectively (Mol. Ther. 1919-1928 (2009)). In parallel batches a transformation frequency of approximately 3.6×10 −6  was determined for the vectors according to the invention which accordingly was still under the value determined for lentiviral vectors. In addition to the transformation frequency the viability and fitness of the transformed cells, respectively, is analysed in this test. The value for this fitness for 3 of 4 so far isolated cells transformed by the vector according to the invention was approximately 10-fold lower than the average value for lentivirally transformed cells. These results by way of the low transformation frequency and the low fitness of cells transformed by the α-retroviral vector according to the invention show that this vector provides a better safety against the oncogene activation than the previous γ-retroviral or lentiviral vectors. The risk of insertional transformation can be reduced further by the choice of suitable internal promoter sequences. 
     Example 4 
     Determination of Deletions of the 3′ U3 Region Eliminating its Promoter and Enhancer Activities 
     The deletion and the substitution of nucleotides of the U3 region, respectively, for the reduction and elimination of the promoter and enhancer activities, respectively, preferably is determined by means of an activity test in which the expression of a reporter gene is measured which is functionally arranged in 3′ to the U3 region provided with deletions. If the expression of a reporter gene which is arranged functionally in 3′ to the deleted and substituted U3 region, respectively, is determined on the level of the background activity of the promoterless expressed reporter gene, the promoter activity according to the invention is eliminated. Preferably, a polyadenylation sequence is arranged in 3′ to the reporter gene. As background activity of the expressed reporter gene e.g. that activity is defined which is measured without any U3 region and without any promoter in 5′ of the reporter gene, respectively. 
       FIG. 14  schematically sectionally shows the nucleic acid constructs with which the activity test for the promoter and enhancer activities of the U3 region as deleted and substituted according to the invention, respectively, was carried out. The construct depicted in the upper part of  FIG. 13  shows the luciferase gene from Renilla (RLuc) as a reporter gene which is flanked in 3′ by a polyadenylation signal sequence of SV40 (SV40 pA) and in 5′ by the nucleic acid sequence to be tested for promoter sequence and enhancer sequence, presently by the wild-type LTR (U3-R-U5) of ASLV. Below that, an otherwise identical construct having a deletion (ΔLTR) is shown, the U3 region of which is partially deleted (ΔU3). In this construct the nucleotides 28-186 are deleted. Further below, an otherwise identical construct is shown in which the LTR is completely substituted by a random sequence (spacer, placeholder derived from the prokaryotic β-lactamase gene). A synthetic polyadenylation signal (syn pA) is arranged in 5′ to the viral sequence section. 
     In  FIG. 15  the luciferase activities of 293T cells are shown which are transfected by the constructs of  FIG. 13  and cultivated under identical conditions. The measurement of the luciferase activity was made in cell homogenates, the depiction shows relative luminescence values which were normalized to total protein and to transfection efficiency. It becomes clear that the deletion to ΔU3 (ASLV delta U3) according to the invention causes a drastic reduction (approximately by the factor 400) of the promoter activity in comparison to the wild-type LTR (ASLV U3). The data after statistical analysis are very significant to each other (**, P&lt;0.01). The substitution of the LTR by the arbitrary sequence (spacer) leads to a similar reduction of the promoter activity. Therefore, according to the invention an arbitrary sequence having no known promoter and enhancer functions can be used as a sequence having background activity. It is assumed that the background activity of the luciferase which is still measured is caused by other components of the plasmid in which the expression cassette of the reporter gene is contained. Preferably, the reporter gene is adapted to the codon usage of the transfected cell.