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Definitions

• the present invention relates to the production of infectious recombinant retrovirus that can be used for the establishment of a stable expression of a gene in eukaryotic cells, for instance for the purpose of human gene therapy.
• a retrovirus is an enveloped RNA virus which carry a reverse transcriptase, which converts its genome into dsDNA and further an intergrase, which catalyses the insertion of the genome into the DNA of host chromosomes (Luciw and Leung 1992).
• the integrated viral genome, or provirus is copied by transcription into RNA molecules that are transported into the cytoplasm and then encapsidated into virus particles.
• the proviral DNA is engineered and transfected into packaging cells (Miller and Rosman 1989; Hodgson 1996).
• the engeneered provirus is called the retrovirus DNA vector. It represents a recombinant retrovirus genome. In this DNA most or all of the gene regions encoding the retrovirus structure proteins .and enzymes have been replaced with a foreign gene.
• the packaging cells represent a stably transformed cell line that produces the retrovirus structural proteins (e.g. the capsid protein gag and the membrane protein env) and enzymes (Miller 1990).
• the retrovirus DNA vector is tranduced into the packaging cells, e.g. by transfection, it will be transcribed by the nuclear transcription machinery into RNA. This RNA is equivalent to the viral RNA. It is called the recombinant retroviral RNA or retrovirus RNA vector.
• the retroviral RNA is transported from the nucleus of the cell to the cytoplasm. In here it will be packaged into a recombinant retrovirus particle , that is a retrovirus vector, through the recognition of its encapsidation signal (a certain RNA sequence) by the virus structural proteins.
• the retroviral vectors are able to infect target cells and facilitate recombinant genome integration into the chromosomes.
• the recombinant genome cannot express viral structural proteins for particle production. It can only express the foreign gene. In this way the recombinant retrovirus can be used as a vector for the expression of a foreign gene for instance in human cells.
• retrovirus vectors Today most retrovirus vectors are based on the Moloney Mouse Leukemia Virus (MLV) but vectors based on the Human Immunodeficiency Virus (HIV)-l has also been developed (Miller and Rosman 1989; Naldini, Blomer et al. 1996; Zufferey, Nagy et al. 1997). While the principle of using retrovirus based vectors for human gene therapy has been proven experimentally, its practical use is still combined with many problems.
• MMV Moloney Mouse Leukemia Virus
• HAV Human Immunodeficiency Virus
• the other problems of the contemporary retrovirus vector systems are related to the low synthesis rate of the viral structural proteins in producer cells, the features of the retrovirus assembly process and the functions of the retroviral structural proteins.
• the cell-targeting function of the vector might be changed by engeneering of the env protein.
• this requires however the construction and testing of many different vector variants and hence also fast and convenient systems to produce retrovirus vectors. The establishment of whole series of different packaging cell lines for such purposes would be extremely time consuming.
• transient production systems of recombinant retrovirus particles have recently been developed (Landau and Littm.an 1992; Soneoka, Cannon et al. 1995).
• the genes for the retrovirus structural proteins and the retrovirus recombinant genome are cotransfected into cells and recombinant retrovirus particles are produced as a result of transient nuclear coexpression of the recombinant retrovirus RNA and the mRNAs for the viral structural proteins and enzymes.
• Using these systems only about three days are required to make a preparation of recombinant retrovirus vectors. However the yield of vectors obtained by these systems is usually very low, especially if a three component gene mixture (the env gene, the gag-pol gene and the recombinant retrovirus DNA) is used for transfection.
• the present invention provides alphavirus-retrovirus RNA-vectors which drive efficient production of infectious recombinant retrovirus particles when introduced into cell cytoplasm of eukaryotic cells.
• a preparation with a high concentration of recombinant retrovirus vectors can be produced by only 10 hr incubation of producer cells.
• genes with introns, and other control elements of gene expression, can be encapsidated into the recombinant particles.
• the vectors are based on the genomic RNA molecule of an alphavirus. These RNA molecules are of plus (+) polarity and translated into the viral polymerase proteins at the onset of alphavirus infection.
• the polymerase replicates the viral genome and also transcribes its 3' end into the viral subgenome that functions as mRNA for the alphavirus structural proteins.
• the alphavirus expression is very efficient and leads to massproduction of viral RNA and proteins. Because of these properties, the alphavirus has been developed into "self-replicating" RNA- vectors for expression of foreign genes in eukaryotic cells (Xiong, Levis et al. 1989; Liljestrom and Garoff 1991). In these vectors the foreign gene is inserted into the subgenomic region of the alphavirus. When the recombinant RNA is transfected into cell cytoplasm, it will be replicated and transcribed into recombinant subgenomes which will be translated into the foreign gene product.
• the recombinant alphavirus genomes can also be packaged into alphavirus particles and transduced into cells by virus infection.
• the recombinant particles are produced by coexpressing the recombinant alphavirus genome together with a "helper" variant of the alphavirus genome.
• the latter contains the complete alphavirus subgenome and its promoter region as well as all of the RNA elements which are required for RNA replication. However, it lacks RNA elements required for packaging.
• the major advantages with the alphavirus expression system are high level expression, fast and convenient usage, and the possibility to use the alphavirus particles to infect a wide range of host cells.
• alphavirus-retrovirus RNA molecules also called alphavirus-retrovirus RNA-vectors
• alphavirus-retrovirus RNA-vectors also called alphavirus-retrovirus RNA-vectors
• retrovirus vectors infectious recombinant retrovirus particles also called retrovirus vectors.
• recombinant alphavirus particles containing aforementioned alphavirus-retrovirus RNA molecules are also called retrovirus vectors.
• the present invention relates to vectors comprising alphavirus RNA having inserted therein a recombinant retrovirus genome.
• the alphavirus RNA comprises a Semliki Forest virus (SFV) RNA and the recombinant retrovirus genome comprises a recombinant genome.
• SFV Semliki Forest virus
• alphavirus RNA with an inserted recombinant retrovirus genome, containing a foreign gene with or without an intron (or some other control element for gene expression), is provided, which permit replication and packaging of said RNA into alphavirus particles in the presence of replication competent helper RNA, which encodes the structural proteins of the alphavirus.
• alphavirus RNA with an inserted recombinant retrovirus genome containing a foreign gene with or without an intron (or some other control element for gene expression), is provided, which permit replication of the said retrovirus genome and its packaging into recombinant retrovirus particles in the presence of replication competent helper RNAs, which encode the retrovirus structural proteins.
• genetically altered alphaviruses and/or cells comprising alphavirus RNA having inserted therein a recombinant retrovirus genome, containing a foreign gene with or without an intron (or some other control element for gene expression), is provided.
• Fig.1 A depicts the DNA sequence near the SFV subgenomic promoter.
• the MLV recombinant genome is inserted into the Sma I site of pSFVl-Nru I vector.
• Fig.l B depicts the pSFVl/LN3i construct. Only the SFV recombinant region of the construct is shown. This region extends from the SP6 promoter (open arrow) to the Nru I site.
• the construct contains, in 5' to 3' direction, (i) the 5' replication signals of SFV RNA, (ii) genes encoding the SFV replication complex (nonstructural proteins, nsp, 1-4), (iii) the internal subgenomic promoter of SFV (solid arrow), (iv) the recombinant MLV genome, including the
• Fig.2 depicts the construction of plasmid pSFVl/LN3i. Relevant restriction endonuclease sites and engineering steps are indicated.
• Fig.3 depicts RNA analysis of transfected cells.
• BHK-21 cells were tr.ansfected with SFVl/LN3i RNA (Lane 1), SFVl/gag-pol RNA (Lane 2), SFV1 Pr80env RNA (Lane 3), or all three RNAs (L ⁇ ine 4).
• Tr.ansfected cells were labeled with [ 14 C]uridine for 6 hr in the presence of actinomycin D.
• Cellular RNAs were isolated and separated on 0.7% agarose gels containing formaldehyde. Radiolabeled bands were visualized by autoradiography. The positions of the replicated genomic and the transcribed subgenomic RNAs are indicated.
• Fig.4 depicts the cell-associated and extracellular protein analysis from BHK-21 cells cotransfected with SFVl/LN3i RNA, SFVl/gag-pol RNA, SFVl/AMenv RNA.
• lys cell lysate
• ip cell lysate immunoprecipitation
• med medium
• M marker.
• the envelope protein products and gag protein products are indicated.
• Fig.5 depicts that the gag precursor production is more efficient in cells transfected with SFV- C/gag-pol RNA than in cells transfected with SFVl/gag-pol RNA.
• the MLV specific Pr65gag, p30 and ⁇ pl2 proteins are indicated.
• Fig.6 depicts the construction of pSFVl/LN-U3insert.
• the recombinant MLV genome US-R-US-yz+'neoR-US-R
• pLN Miller and Rosman 1989
• 35-base SFV sequence (denoted with *) which is flanking the recombinant MLV genome on its 5' side and which contains part of the SFV subgenomic promoter is also inserted into 3' U3 just after the sequence specificing for DNA integration (Fig. 6 B and C).
• Fig.7 depicts the construction of the plasmid pSFVl-I-CAT.
• A is a schematic representation of the structure of the pCAT3-promoter vector. The engineering strategy of pSFVl-I-CAT is shown in (B).
• C is a schematic representation of the recombined SFV region of SFV1-I-CAT.
• the CAT gene with the intron was isolated from the pCAT3-promoter vector (Promega) (Fig. 7A) and inserted as a Bgl II-Bam HI fragment into an pSFVl/LN3i. To facilitate this, a unique Bam HI site was created into the latter plasmid at a position after the neo R gene region.
• pSFVl/LN3i The intermediate was denoted pSFVl/LN3i (BNNP). This required first the removal of two existing Bam HI sites followed by the insertion of a new site.
• Fig. 7B shows schematically the engineering strategy and Fig. 3C the functional gene regions of the recombined SFV part in pSFVl-I-CAT.
• the intronless pSFVl-CAT was derived from pSFVl-I-CAT by excising the intron containing DNA fragment with Hind HI.
• Fig.8 depicts CAT activity in NIH 3T3 cells infected with recombinant retrovirus particles containing the Cat gene with (SFV1-I-CAT) or without intron (SFV 1 -CAT).
• NIH 3T3 cells were plated into 60 mm dishes at 5 x 10 5 cells/dish 24 hr before infection. The cells were infected with 1 x 10 5 recombinant retrovirus particles.
• CAT activity was tested using CAT Enzyme Assay System With Reporter Lysis Buffer (Promega) 52 hr after infection.
• the cells extracts were incubated in a reaction mix containing [ 14 C]labeled chloramphenicol and n- Butyryl Coenzyme A at 37°C for 16 hr.
• CAT transfers the n-butyryl moiety of the cofactor to chloramphenicol.
• the reaction products were extracted with 300 ⁇ l of xylene.
• the n-butyryl chloramphenicol partitions mainly into the xylene phase, while unmodified chloramphenicol remains prodominantly in the aqueous phase.
• the xylene phase was mixed with 3ml scintillant and counted in the liquid scintillation counter.
• the cpm measured in each sample represents the butyrylated chloramphenicol.
• RNA vectors of the present invention provide a means for replicating and expressing recombinant retrovirus genomes independent of the host nucleus in the cytoplasm of several different types of eukaryotic cells.
• the expressed recombinant retrovirus genomes can be packaged into infectious recombinant retrovirus particles, also referred to as "retrovirus vectors", if coexpressed with retrovirus structural proteins.
• the recombinant retrovirus genome refers to a "retrovirus RNA genome” that contains all those RNA elements that are required in cis for retrovirus genome encapsidation into retrovirus p.articles, reverse transcription, dsDNA synthesis, retrovirus DNA integration into host chromosomes and retrovirus DNA transcription in cell nucleus.
• the recombin.ant retrovirus particle refers to a particle in which a recombinant retrovirus genome (with or without an intron or some other control elements of gene expression) has been packaged into a retro virus-like particle. This recombinant particle can mediate the transduction of the recombinant retrovirus genome into a new cell via the process of retrovirus infection.
• RNA vector we used the pSFVl-Nrul plasmid into which we inserted the recombinant MLV genome, R-
• the pSFVl-Nrul plasmid corresponds to the earlier described pSFVl plasmid (Liljestrom and Garoff 1991), but it contains a 527 base pair deletion between the Stu I and Hind in sites of pSFVl and furthermore the Spe I site of pSFVl has been changed into Nru I site.
• the insertion of the recombinant retrovirus genome into pSFVl-Nru I was made so that the recombinant retrovirus genome followed in 3' direction the promoter region for the SFV subgenome (Fig. 1 A).
• the recombinant retrovirus genome is expressed instead of the viral subgenomic RNA.
• the promoter region for the SFV subgenome overlaps with the extreme 5' region of the subgenomic transcript itself, the 5' end of the recombinant retrovirus gene cannot be joined directiy at the transcription start site, but somewhat further down-stream (Strauss and Strauss 1994).
• the transcribed recombinant retrovirus genome will contain 38-base SFV specific residues at its 5' end (see Fig.lA).
• the recombinant retrovirus genome (U3-R-U5- ⁇ / + -neo R -U3-R) from pLN was inserted between the BamH I and Sma I sites of pSFVl-Nru I plasmid (see Fig. 6A and B).
• a 35-base SFV specific sequence which contains part of the SFV subgenome promoter region is located between the SFV transcription initation site and the start of the recombinant retrovirus genome.
• the same sequence was also inserted into the 3' U3 region of the recombinant retrovirus genome at a position just downstream of the region specifying for retrovirus DNA integration (Fig. 6B and C).
• the double-stranded DNA synthesis process of the recombinant retrovirus will result in a DNA molecule that can be integrated into chromosomes so that no SFV specific sequences will be present in its 5' end and also no SFV sequences will be present in the 5' end of transcribed RNA.
• Plasmid pSFVl/LN3i was used for transcription of corresponding RNA vector in vitro. This was done as described using SP6 polymerase (Liljestr ⁇ m and Garoff 1991). The RNA was transfected into BHK-21 cells and its replication in cell cytoplasm was followed by labeling with 14 C-uridine for 6 hr. Samples containing cytoplasmic RNA were analysed on a agarose gel. Fig.3, lane 1, shows that both full-sized and subgenomic RNA have been produced. This indicates that the SFVl/LN3i RNA vector can be used for the production of recombinant retrovirus genome molecules in cell cytoplasm.
• RNA transfection for introducing the vector RNA into cell cytoplasm.
• An alternative method is to package this RNA into SFV particles using cotr.ansfection with SFV-helperl RNA (Liljestr ⁇ m and Garoff 1991) and then to infect the BHK-21 cells with the recombinant SFV particles.
• RNAs were transcribed form the plasmids pSFV-C/gag-pol (or pSFVl/gag-pol) and pSFVl/AMenv (or pSFVl/Pr80env).
• the pSFVl/gag-pol plasmid contains the coding-region of the gag-pol of MLV (Suomalainen .and Garoff 1994). This is also present in pSFV-C/gag- pol. The latter plasmid contains in addition the SFV capsid coding-region in front of gag-pol. In the transcribed C-gag-pol RNA, the C-region specifies increased translation efficiency as compared to gag-pol mRNA (Sj ⁇ berg, Suomalainen et al. 1994).
• the pSFVl/Pr80env contains the coding region of the homologous (ecotropic) MLV env precursor protein, and the SFVl/AMenv contains the coding region of the heterologous amphotropic env precursor protein.
• the latter envelope protein has the capacity to target the recombinant retrovirus particle to a broad range of animal host cells including human cells, whereas the ecotropic env only recognizes mouse cells.
• the protein synthesis in cells cotransfected with SFVl/LN3i, SFV- C/gag-pol and SFVl/AMenv RNAs was followed by metabolic labelling with [ S]methionine. The results of a pulse-labelling experiment is shown in Fig.4.
• RNA made from pSFVl/LN-U3insert Similar studies were performed with RNA made from pSFVl/LN-U3insert. The titer of the corresponding recombinant retrovirus preparation was approximately the same as that one which was obtained with the RNA from pSFVl-LN3i.
• RNA was then transfected into BHK-21 cells together with the SFV-C/gag-pol and the SFV 1 -env RNAs. The latter two RNAs specifed gag-pol and env precursor production. The cells were incubated for 10-15 h after transfection and the media was collected. The released recombinant retroviruses were then used to transduce CAT genes into NIH 3T3 cells. The CAT activity of cells was measured using a standard CAT assay after 52 hours (Fig.8). Very high CAT activity was found in the cells infected with vectors containing CAT gene with the intron whereas very low activity was found in the cells transfected with the intronless vector. Thus, this shows that the intron containing CAT gene was successfully transduced with the retrovirus vector into the recipient cells and that it resulted in efficient CAT expression.
• replication competent retrovirus particles A replication competent particle has aquired all retrovirus structural protein genes and hence it has the capacity to spread from cell to cell. Such particles can be generated in the producer cell through the process of RNA recombination.
• the possible generation of replication-competent particles in our production system was tested using a marker rescue assay (van Beusechem, Kukler et al. 1990). No replication-competent particles were found in a sample containing 2.6 x 10 6 infectious recombinant particles.
• alphavirus-retrovirus RNA vectors can be used for the expression of a recombinant retrovirus genome which can be packaged into infectious recombinant retrovirus vectors carrying either the amphotropic or ecotropic envelope proteins without detectable production of any replication competent particles.
• ⁇ -globin gene complex including an intron and certain locus control elements (Chang, Liu et al. 1992). With our sytem it should be possible to package this gene complex into retrovirus vectors at high titer and use them for the treatment of hemoglobin disorders like ⁇ -thalassemia.
• factor IX gene-intron complex has been characterized that direct efficient factor IX expression (Kurachi, Hitomi et al. 1995). This should also be possible to package into retrovirus vectors using the system we have described in this disclosure. Such vectors could be useful for gene therapy of patients suffering from bleeding disorder hemophilia B (Christmas disease).
• retrovirus vector production system is very fast and efficient: only 10 hr incubation of transfected cells is required to produce a preparation which contains a high concentration of vector particles (>10 6 particles/ml).
• the system allows for the convenient variation of the qualities of the packaging components and hence also the functions of the recombinant retrovirus particles. Therefore, this new retrovirus vector production system should meet the need for an efficient, fast and convenient production system of recombinant retrovirus particles. Its use should speed-up the engeneering of particles that are more suitable for specific gene therapy purposes.
• SFV expression vectors for production of MLV vectors. Because of the great similarities among the various alphaviruses (Strauss and Strauss 1994) it is expected that any alphavirus expression vector (e.g. a Sindbis virus vector, (Xiong, Levis et al. 1989)) can be used for the production of a retrovirus vector. Similarly we have in our examples only shown how to produce MLV vectors using alphavirus vectors but it should be equally possible to use our system for the production of other retrovirus based vectors e.g. HIV-1 vectors (Naldini, Bl ⁇ mer et al. 1996; Zufferey, Nagy et al. 1997).
• retrovirus based vectors e.g. HIV-1 vectors (Naldini, Bl ⁇ mer et al. 1996; Zufferey, Nagy et al. 1997).
• primer A 5' GCTCTAGAGAACCATCAGATG 3' (21 mer)
• primerB 5'GGGGATCCAATCAGAATTCTGTGTATTAACGCACCAAT
• primer D 5' CCCAAGCTTTGCAACTGCAAGAGGGTTTA 3' (29 mer)
• the pLN EcoR I fragments containing the 3'LTR were used as the template DNA.
• the reaction mixture was denatured at 94°C for 45 s, annealed ar 50°C for 45 s, and elongated at 72°C for 1 min. After 25 cycles of amplification, the fusion PCR products were purified using Wizard PCR Preps DNA Purification System (Promega, SDS, Falkenberg, Sweden).
• the fusion PCR fragment was digested with Hind III and Xba I, and subcloned between Hind III and Xba I sites of pUC18 plasmid vector, making pUC18/insert plasmid.
• the fusion PCR fragment was verified by sequence analysis.
• the pUC18/insert plasmid was cut with Hind III, filled with DNA polymerase I large (Klenow) fragment and then cut with Xba I.
• a 262 bp Hind III (blunt) - Xba I fragment was isolated.
• the pLN plasmid was cut with Asc I, filled with DNA polymerase I large (Klenow) fragment and then cut with Xba I.
• the 2221 bp Asc I (blunt) - Xba I fragment was isolated.
• the pSFVl/LN3i was made by ligating the pLN/Asc I (blunt) - Xba I fragment and pUC18/insert Hind III (blunt) - Xba I fragment into Sma I cut of pSFVl- Nru I, as shown in Fig.2.
• Plasmid pSFVl/gag-pol contains the coding sequence of MLV retroviral structural precursor protein gag and the fusion protein gag-pol. The pol-part of the latter is the precursor for all viral enzymes.
• plasmid pSFV-C/gag-pol the translation enhancing RNA sequence of the SFV capsid gene was inserted in front of the gag-pol gene in pSFVl/gag-pol.
• pSFVl/gag-pol was made by inserting the MLV gag-pol cDNA from pNCA (Colicelli and Goff 1988) into the BamEl site of pSFVl.
• the two Spe I sites in the gag-pol cDNA were removed by site-directed mutagenesis, using the oligonucleotide 5'GGGGGGTTGTTTGACGAGTGCCTCTACTGCATGGGGGG CCAGAATGACGAGTGGCTGTCCCATGGT 3' (Su and El-Gewely 1988).
• pSFV-C/gag-pol was made by ligating the Not I - Bsm I fragment (14410 bp) of pSFV-1 /gag-pol and the Not I - Bsm I fragment (2723 bp) of pSFV-C/Pr65gag (Suomalainen and Garoff 1994).
• Plasmid pSFVl/AMenv contains the coding sequence of the murine a photropic virus (4070A) envelope protein.
• the amphotropic envelope gene fragment from pPAM3 was first inserted into pUC18 by subcloning steps to make pUC18/AMenv.
• the plasmid pSFVl/AMenv was made by inserting the Sma I- Hpa I fragment (1976bp) from pUC18/AMenv into the Sma I site of pSFVl-Nru I.
• Plasmid pSFVl [Liljestr ⁇ m, 1991 #15] was cleaved with Stu I and H d III and the large fragment was filled with DNA polymerase I large (Klenow) fragment and ligated. The deleted plasmid molecule was cloned and used for in vitro mutagenesis. In this step, the Spe I recognization sequence (ACTAGT) was changed to that of Nru I (TCGCGA). This created the plasmid pSFVl-Nru I.
• RNA (20 ⁇ l) was transfected into 8 x 10 6 B ⁇ K-21 cells (American Type Culture Collection, Rockville, Maryland,USA) by electroporation. Electroporation was carried out at room temperature by two consecutive pulses at 0.85 kV and 25 ⁇ F, using Bio-Rad Gene Pulser apparatus (Richmond, California, USA).
• Transfected B ⁇ K-21 cells were plated onto 33mm culture dishes and incubated for 2 hr at 37°C. Media were removed and replaced with 1 ml aliquots of medium containing l ⁇ g/ml actinomycin D (Sigma-Aldrich Sweden, Sweden). After incubation for 2 hr at 37°C, media were replaced with 1 ml aliquots of medium containing l ⁇ g/ml actinomycin D and 75 Kbq [ 1 C]uridine (2.1GBq/mmol, DuPont, Du Medical Scandinavia AB, Sollentuna, Sweden).
• SFVl/LN3i RNA and other two RNAs which contain the coding region of retrovirus gag-pol and env respectively, all of the genomic and subgenomic RNAs were produced in the cotr.ansfected cells (Fig.3, lane 4).
• Lanes 2 and 3 show RNA production in cells transfected with SFVl/gag-pol RNA and SFV1/Pr80env RNA, respectively.
• This example demonstrates viral protein synthesis in cells cotransfected with SFVl/LN3i RNA, SFVl/gag-pol RNA and SFVl/AMenv RNA by electroporation.
• Transfected cells were added to 9 ml complete BHK-21 medium, plated onto three 33-mm culture dishes and incubated at 37°C.
• PBS phosphate- buffered saline
• transfected cells were washed twice with phosphate- buffered saline (PBS) and starved by incubation at 37°C for 30 min in 2 ml methionine-free minimum essential medium (MEM, GLBCO, Life Technologies AB, Taby, Sweden) supplemented with 20mM Hepes.
• MEM methionine-free minimum essential medium
• Extracellular particles in media samples were pelleted through a 20% sucrose cushion (17,000 rpm, 2 hr, 10°C, Beckman JA18.1 rotor). Pellets were analyzed by SDS-PAGE as described above. Gels were dried and exposed to Fuji film (Fuji Photo Film Co., LTD., Tokyo, Japan). The results are shown in Fig.4. All of the retrovirus proteins were synthesized in trasfected cells and incoporated into virus-like particles. The gag precursor protein (Pr65) was observed in cell lysate and virus-like particles. Most of the Pr65 was cleaved into the mature products, p30 and ppl2.
• plO nucleocapsid protein
• the amphotropic envelope precursor protein (Pr85) in cell lysate was cleaved into surface proteins (gp70) and transmembrane proteins (pl5E) by cellular protease. Only gp70 and pl5E were incoporated into virus-like particles. The pl5E was cleaved into pl2E by viral protease.
• This example demonstrates that the expression of gag-pol products in the cells transfected with SFV-C/gag-pol RNA is much higher than in the cells transfected with SFVl/gag-pol RNA.
• BHK-21 cells were transfected with 20 ⁇ l of SFV-C/gag-pol RNA or 20 ⁇ l of SFVl/gag-pol RNA by electroporation. The transfected cells were pulsed for 30 min and chased for 15 min to 2 hr as decribed above. The cell-associated and extracellular MLV proteins were analyzed by SDS-PAGE(12%) under reducing condition. The results are shown in Fig.5. About 5-fold more gag-pol products were produced in the cells tr.ansfected with SFV-C/gag-pol RNA, as compared with that were produce in the cells transfected with SFVl/gag-pol RNA.
• This example demonstrates that infectious recombinant retrovirus particles is produced by cells cotransfected with SFVl/LN3i RNA, SFVl/gag-pol RNA (or SFV-C/gag-pol RNA), and SFV1/Pe80env RNA (or SFVl/AMenv RNA).
• the transfected BHK-21 cells were diluted into 9 ml complete BHK medium, and 6 ml of the cell suspension (containing 4 x 10 6 living cells) was plated onto a 60-mm culture dish (Nunclon, Roskilde, Denmark). The cells were incubated at 37°C, and the media were harvested at 5 hr interval from the same dish and replaced with 2 ml aliquots of fresh complete BHK-medium. The media were passed through a 0.45 ⁇ m filter
• Ne ⁇ R -transduction-competent retrovirus particles were titrated on NIH 3T3 cells. Therefore, NLH 3T3 cells were seeded at 5 x 10 5 cells per dish (60-mm) on day one.
• NLH 3T3 cells were seeded at 5 x 10 5 cells per dish (60-mm) on day one.
• 1 ml aliquots of 10-fold serial dilutions of media samples were added to cell monolayers in the presence of 4 ⁇ g/ml Polybrene (Sigma-Aldrich Sweden, Sweden). After incubation for 2 hr at 37°C, 1 ml .aliquots of medium containing 4 ⁇ g/ml Polybrene was added to each dish, and incubation was continued at 37°C.
• Virus titers are given as colony-forming units per ml (cfu ml). They were calculated by multiplying the number of colonies with the dilution times and divided by 2 to account for cell doubling. Table 1. Release of infectious recombinant retrovirus particles from transfected BHK-21 cells'
• H3T3 cells were incubated with diluted medium of transfected BHK-21 cells and then subjected to G418 selection.
• the numbers refer to resistant colonies formed after 12 days incubation.
• gag-pol products in cells transfected with SFV-C/gag-pol RNA is much higher than that of the corresponding products in SFVl/gag-pol RNA transfected cells.
• SFV-C/gag-pol RNA was used in a cotransfection/time course experiment, the production of infectious particles was considerably increased.
• the titer in most 5 hr-media samples was about 4 x 10 6 CFU/ml.
• Example 10 This example demonstrates that replication-competent particles were not detected.
• the possible presence of replication-competent particles in supernatant media was tested by a rescue assay.
• 3T3Zip «eoSV(X)p cells, an NIH 3T3-derived cell line that harbours recombinant provirus consisting of the MLV LTRs, a packaging signal and the neo R -gene were utilized in this assay: Transfection of these cells by the genes encoding the MLV gag-pol- and env-proteins results in the production of infectious particles containing the neo R -recombinant genome.
• 3T3ZipneoSV(X)p cells were infected with the supernatant medium containing 2.6 x 10 6 infectious recombinant retrovirus particles in the presence of 4 ⁇ g/ml Polybrene.
• the infected cells were passaged for 8 days. When the cells were about 50% confluent, the medium was replaced with fresh medium and the cells were incubated at 37°C. After a 24 hr incubation, the medium was collected, passed through 0.45 ⁇ m filter and analyzed for the presence of neo R - transduction-competent particles by titration on NTH 3T3 cells as described above.
• pSFVl/LN-U3insert contains the recombinant retrovirus genome, US-R-US-i/ ⁇ -neo ⁇ -US-R in the SFV subgenome region (Fig. 6). This was done as follows: (1) A 464 bp Sfc I - Kpn I fragment from the 3'LTR of pLN was cloned between Bgl ⁇ and Kpn I sites of pSP73, to make pSP73/U3. The Sfc I .and Bgl II ends were filled with Klenow fragment.
• Primers used for fusion PCR were upper 5' TGCTTGCCGAATATCATGGTG 3', lower primer 5' CCCAAGCTTTGCAACTGCAAGA GGGTTTA 3', and fusion primers 5' GATCCAATCAGAATTCTGTGTATTAACGCACCA ATGGTGGGGTCTTTCATTCCCC 3', 5' ATTGGTGCGTTAATACACAGAATTCTGATT GGATCTGTAGGTTTGGCAAGCTAGC 3'.
• the PCR reaction were carried out at 94°C for 45 s, 60°C for 45 s, and 78°C 2 min using the Nco I - Nde I fragments as the template DNA.
• the 862 bp fusion fragment s were purified using Wizard PCR Preps DNA Purification System (Promega, SDS, Falkenberg, Sweden). (4) The fusion PCR fragment was cut with NgoM I and Hind HI and inserted between NgoM I and Hind III sites of pSP73/LN, to make pSP73/LN-U3insert. (5) pSP73/LN-U3 insert was cut with Hind III, filling the end with Klenow fragment, and then cut with Bgl II. The 2973 bp Bgl II - Hind III (blunt) fragment was isolated.
• the pSFVl/LN-U3insert was made by inserting the Bgl II - Hind III (blunt) fragment of pSP73/LN-U3insert between the BamH I and Sma I sites of pSFVl-Nru I.
• This example describes the construction of pSFVl ⁇ N3i (BNNP).
• the plasmid was derived from pSFVl/LN3i by removing the two existing Bam Hi sites and including a group of unique sites, also BamH I.
• the BamH I sites were removed by cutting pSFVl/LN3i with BamH I, filling with Klenow fragment, and religating.
• the resulting plasmid was called pSFVl/LN3i (- B).
• the group of new sites was inserted by fusion PCR.
• the sites included BamH I, Nde I, Nsi I and Pme I.
• Primers for fusion PCR were: 5' TGT CAA GAC CGA CCT GTC GC 3' (primer 1), 5' CCC AAG CTT TGC AAC TGC AAG AGG GTT TA 3' (primer 2), 5' GGA TCC ATA TGC ATG TTT AAA CGG ACT CTG GGG TTC GAT AAA 3' (primer 3) and GTT TAA ACA TGC ATA TGG ATC CCG CTC AGA AGA ACT CGT CAA 3' (primer 4).
• pSFVl/LN3i (-B). With the first two primers a 678 bp fragment containing the 3' end of the neo R gene was synthesized.
• primers 3 and 4 we synthesized a partial overlapping 641bp fragment containing the 3' LTR.
• the fusion PCR reaction resulted in a 1297 fusion fragment containing the unique sites. This was cut with BssH 2 and the 747 bp fragment isolated and inserted into BssH 2 cut pSFVl/LN3i (-B).
• the resulting plasmid was called pSFVl/LN3i(BNNP).
• a CAT gene fragment plus an intron was isolated from pCAT3 ® -promoter vector (Promega, Catalog #E1861) by cleavages with Bgl II and Bam HI.
• the 1389 bp fragment was purified and inserted into pSFVl/LN3i(BNNP). This was done in a two fragment ligation with Bam H 1 CAT and dephosphorylated pSFVl/LN3i (BNNP).
• the resulting plasmid was called pSFVl-I- CAT.
• the pSFVl-CAT was done similarly using the pCAT3 ® -promoter vector from which the intron had been removed. This was done by cleaving the latter plasmid with Hind III.
• Retrovirus vectors containing the CAT gene with or without the intron were produced by cotransfection of SFVl-I-CAT RNA or SFV 1 -CAT RNA with both SFV-C/gag- pol RNA and SFV 1 -env RNA into BHK cells. After incubation for 10-15 h media were collected and used for titration of neo R transduction competent particles. The titers were about 4xl0 5 particles/ml, for SFVl-I-CAT and lxlO 6 particles/ml for SFV1-CAT.
• Example 15 CAT expression efficiencies in cells transduced with recombinant retrovirus particles containing a CAT gene with and without an intron.
• About lxlO 6 cells were infected with lxlO 5 recombinant retrovirus particles.
• After 52 h lysates were prepared and CAT activity measured by using a standard assay (CAT Enzyme Assay System With Reporter Lysis Buffer, Promega).
• the results showed about 30 fold higher CAT activity in cells transduced with recombinant retrovirus particles containing CAT with an intron (Fig.8).
• this example shows that an intron containing gene can be transduced into cells with our recombinant retrovirus particles and that this results in improved expression.

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