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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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  • C12N2740/00—Reverse transcribing RNA viruses
  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
  • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
  • C12N2740/13043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

• This invention relates to retroviral vectors. More particularly, this invention relates to
• retroviral vectors having multiple cloning, or restriction enzyme recognition sites, and to systems for the exchange of gene sequences between vectors having compatible or complementary multiple cloning sites.
• Retroviral vectors are useful as agents to mediate retroviral-mediated gene transfer into eukaryotic cells. Such vectors are generally
• Retroviral vectors have also been constructed which can introduce more than one gene into target cells. Usually, in such vectors one gene is under the regulatory control of the viral LTR, while the second gene is expressed either off a spliced message or is under the regulation of its own, internal promoter.
• a packaging-defective helper virus is necessary to provide the structural genes of a retrovirus, which have been deleted from the vector itself.
• MoMuLV Moloney murine leukemia virus
• pPr80 gag another glycosylated protein
• MoMuSV Moloney murine sarcoma virus
• Safety is derived from the combination of vector genome structure together with the packaging system that is utilized for production of the infectious vector.
• Miller, et al. have developed the combination of the pPAM3 plasmid (the packaging-defective helper genome) for expression of retroviral structural proteins together with the LN vector series to make a vector packaging system where the generation of recombinant wild-type retrovirus is reduced to a minimum through the elimination of nearly all sites of recombination between the vector genome and the packaging-defective helper genome (i.e. LN with pPAM3).
• the LN series of vectors has generated a vector backbone which incorporates several safety features, the LN vector contains a very limited number of potential cloning sites for the insertion of additional genes into the vector.
• Gene therapy or drug, delivery via gene transfer entails the creation of specialized vectors each vector being applicable only to a particular disease.
• a vector cloning system be available which consistently maintains the necessary safety features yet permits maximal flexibility in vector design. Subtle changes in gene position, or in the specific combination of regulatory sequence(s) with the gene of interest, can lead to profound differences in vector titer or in the way that transferred genes function in target cells. Current vector designs require that for each combination of genes and promoters, the entire vector be
• retroviral vectors which also permits consistent vector construction, whereby genes, promoters, or combinations of genes and promoters may be rapidly exchanged and the vectors evaluated to achieve optimal results in tissues of interest.
• a retroviral vector which includes at least four cloning, or restriction enzyme recognition sites, wherein at least two of the sites have an average frequency of appearance in eukaryotic genes of less than once in 10,000 base pairs, i.e., the restriction product has an average DMA size of at least 10,000 base pairs.
• Preferred cloning sites are selected from the group consisting of Notl, SnaBI, Sall, and Xhol.
• Such vectors may be engineered from existing retroviral vectors through genetic engineering techniques known in the art such that the resulting retroviral vector includes at least four cloning sites wherein at least two of the cloning sites are selected from the group consisting of the NotI, SnaBI, Sall, and Xhol cloning sites.
• the retroviral vector includes each of the Notl, SnaBI, Sall, and Xhol cloning sites.
• retroviral vectors which may be transformed to include the above-mentioned cloning sites include Moloney Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus and Harvey Sarcoma Virus. Specific vectors which may be constructed in
• Such a retroviral vector may serve as part of a cloning system for the transfer of genes to such retroviral vector.
• a cloning system for the manipulation of genes in a retroviral vector which includes a retroviral vector including multiple cloning sites of the type
• shuttle cloning vector which includes at least two cloning sites which are compatible with at least two cloning sites selected from the group consisting of Notl, SnaBI, Sall, and Xhol located on the retroviral vector.
• the shuttle cloning vector also includes at least one desired gene which is capable of being transferred from said shuttle cloning vector to said retroviral vector.
• the shuttle cloning vector may be constructed from a basic "backbone" vector or fragment to which are ligated one or more linkers which include cloning or restriction enzyme recognition sites . Included in the cloning sites are the compatible, or
• the shuttle cloning vector can be employed to amplify DNA sequences in prokaryotic systems.
• the shuttle cloning vector may be prepared from plasmids generally used in prokaryotic systems and in
• the shuttle cloning vector may be derived from plasmids such as pBr322; pUC 18; etc.
• the present invention provides for retroviral vectors having a larger choice of cloning sites, and shuttle vectors with complementary cloning sites, which provides for the rapid exchange of genes and/or promoters from the shuttle vector to the retroviral vector.
• the increased number of cloning sites also provides for greater flexibility in vector
• Notl, SnaBI, Sall, and Xhol cloning sites are sites which are of extreme rarity in eukaryotic genes. The use of such "rare" sites enables one to extract a first gene from the
• retroviral vector and replace the first gene with a second gene without altering the retroviral vector backbone structure.
• reconstruction of the entire retroviral vector is not necessary.
• the order of the cloning sites in the retroviral and shuttle vectors be
• vectors are constructed which may be efficiently evaluated to achieve optimal results in tissues of interest.
• a retroviral vector said vector including a 5' LTR (long terminal repeat) and a 3' LTR. At least the promoter sequence(s) of the 3' LTR is mutated, or altered, such that the promoter sequence becomes non-functional. Such a mutation, however, does not alter the overall
• the enhancer sequence(s) of the 3' LTR may also be mutated such that the enhancer sequence(s) also becomes non-functional.
• the integrator sequence is maintained.
• the altering or mutating of only the promotar or enhancer sequences of the 3' LTR does not require a large deletion of the 3' LTR to eliminate promoter or enhancer function, and the overall structure of the 3' LTR is preserved.
• LTR structure is critical for efficient integration of the vector into the genome, one is able to maintain a high titar of retroviral vector. Such vectors, therefore, may be useful in clinical applications where the maintenance of a high titer of vector is essential or critical.
• Such a retroviral vector may be formed from a retroviral vector having at least four cloning sites, wherein at least two of the cloning sites are selected from the group consisting of Notl, SnaBI, Sall, and Xhol, as hereinabove described; however, the scope of the present
• vectors whose promoter sequence of the 3' LTR may be mutated include Moloney Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus and Harvey Sarcoma Virus. Such mutations, whereby the promoter or enhancer sequences of 3' LTR are altered, may be effected through techniques known in the art.
• Plasmid pG1 was constructed from pLNSX (Palmer et al., Blood, 73:438-445; 1989). The construction strategy for plasmid pG1 is shown in Figure 1. The 1.6 kb EcoRI fragment, containing the 5' Moloney Sarcoma Virus (MoMuSV) LTR, and the 3.0 kb EcoRI/Clal fragment, containing the 3' LTR, the bacterial origin of replication and the ampicillin resistance gene, were isolated separately. A linker containing seven unique cloning sites was then used to close the MoMuSV LTR, and the 3.0 kb EcoRI/Clal fragment, containing the 3' LTR, the bacterial origin of replication and the ampicillin resistance gene, were isolated separately. A linker containing seven unique cloning sites was then used to close the
• pG1 consists of a
• retroviral vector backbone composed of a 5' portion dervied from MoMuSV, a short portion of gag in which the authentic ATG start codon has been mutated to TAG (Bender et al. 1987), a 54 base pair multiple cloning site (MCS) containing from 5' to 3' the sites EcoRI, Notl, SnaBI, SaLl, BamHI, Xhol, Hindlll, Apal, and Clal, and a 3' portion of MoMuLV from base pairs 7764 to 7813 numbered as described in (Van Beveren et al., Cold Soring Harbor, Vol. 2, pg. 567, 1985). ( Figure 2).
• the MCS was designed to generate a maximum number of unique insertion sites, based on a screen of non-cutting restriction enzymes of the pG1
• the plasmid the neo R gene, the ⁇ -galactosidase gene, the hygromycin R gene, and the SV40 promoter.
• the pGene plasmid ( Figure 3) does not exist as an independent molecular entity, but rather may be considered a construction intermediate in the process of cloning genes for subsequent insertion into pG1.
• the basic backone is that of pBR322 (Bolivar et al., Gene, 2:95 1977).
• pBR322 Bosset plasmid plasmid plasmid plasmid plasmid ( Figure 3) was used as an independent molecular entity, but rather may be considered a construction intermediate in the process of cloning genes for subsequent insertion into pG1.
• the basic backone is that of pBR322 (Bolivar et al., Gene, 2:95 1977).
• To the 2.1 kb EcoRI/Ndel fragment containing the ampicillin resistance gene and the bacterial origin of replication two linkers were ligated. These linkers, synthesized using an
• oligo-nucleotide synthesizer contain a total of 14 unique restriction enzyme recognition sites, as well as sequences felt to enhance mRNA stability and translatability in eukaryotic cells.
• the restriction sites were chosen based on a screen of non-cutting restriction enzymes of the plasmid backbone, the neo R gene, the ⁇ -galactosidase gene, the hygromycin R gene, and the SV40 promoter. Genes can be ligated into this backbone with Ncol and Xhol ends.
• the resulting backbone less the inserted gene, is roughly 2.1 kb in size and contains a 99 base pair multiple cloning site containing, from 5' to 3', the following restriction enzyme recognition sites: Sphl, Notl, SnaBI, Sall, SacII, Accl, Nrul, Bglll, Ncol, Xhol, Hindlll, Apal, and Smal ( Figure 4). From the Bglll to the Ncol sites lies a 27 base pair region containing an mRNA signal based on the work of
• adenovirus 2 late proteins may also follow this rule (Lawrence and Jackson, J. Molec. Biology, 162:317-334(1982)).
• a consensus signal for initiation of translation based on Kozak's rules (Kozak, Nucl. Acids Res., 12:857-872 (1984)) was also inserted.
• the wobble at the ATG was used which permitted use of an Ncol restriction enzyme site.
• genes may be inserted in between the Ncol and Xhol sites. Promoters may then be added by insertion into the Nrul site, if the restriction enzyme map of the inserted gene leaves this site as unique.
• the construction of the multiple cloning site is such that, even if some sites no longer remain unique after a gene is inserted, there is a substantial likelihood that sites 5' of the Bglll site remain available for promoter insertions.
• the first is called pGlN2SvBg, a vector using the bacterial neomycin resistance (neo R ) gene as a selectable marker and also containing the bacterial ⁇ -galactosidase ( ⁇ -gal) gene under regulation of the SV40 early promoter.
• pGlN2SvBg a vector using the bacterial neomycin resistance (neo R ) gene as a selectable marker and also containing the bacterial ⁇ -galactosidase ( ⁇ -gal) gene under regulation of the SV40 early promoter.
• neo R bacterial neomycin resistance
• ⁇ -gal ⁇ -galactosidase
• pBg that is the pGene backbone with the ⁇ -gal gene inserted into the multiple cloning site.
• the 3.0 kb BamHI/EcoRI fragment of the lacZ gena encoding/- ⁇ -galactosidase was isolated and two linkers were added.
• an Ndel-BamHI linker containing the 5' portion of the multiple cloning site up to the Ncol site, as well as the first 21 base pairs of the lacZ gene, was ligated.
• an EcoRI/EcoRI linker completing the 3 ' sequence of the lacZ followed by sequence encoding the Xhol, HindXII, Apal, and Smal sites was ligated.
• the sequence of the 5' EcoRI site was mutated, maintaining amino acid coding fidelity but eliminating the internal EcoRI site to permit
• Vector producer cell lines were prepared using established protocols.
• the packaging cell line PE501 (Miller and Rosman, Biotechniques 7:980-990 (1989)) was plated at a density of 5 x 10 5 cells per 100 mm plate and the following day purified vector DNA was introduced using standard CaPO 4 precipitation (Wigler et al., Cell 14725-731 (1978)).
• 20-40 ⁇ g of vector DNA was prepared with a co-precipitate consisting of 0.25M CaCl 2 /l mM Hepes (pH 7.2) and 140 mM NaCl, 0.75 mM Na 2 HPO 4 , 25 mM Hepes (pH7.2).
• the DNA/precipitate was allowed to sit at room temperature for 30 min and then added (1 ml/plate) to the cells in tissue culture medium (DMEM + 10% fetal Bovine serum) for an overnight .incubation. The medium was changed to fresh DMEM + serum the following morning.
• the transfected cells were allowed to grow to near confluence for the next 48 hours, at which point virus supernatant was collected to infect a separate population of PA317 vector packaging cell lines at a density of 1 x 10 5 cells per 100 mm plate seeded 24 hours prior to infection.
• the standard infection conditions include undiluted virus supernatant, filtered through a 0.2 uM membrane, to which 8 ug/ml polybrene is added.
• the transduced cells can then be analyzed directly based on ⁇ -gal expression or be selected with the neomycin analogue, G418 sulfate, to enrich for cells expressing the neo r gene.
• NIH 3T3 cells were plated at a density of 2 x 10 4 cells per 35 mm dish and the following day infected for 2-4 hours with various dilutions of virus supernatant containing 8 ug/ml polybrene. The cells were allowed to grow for an additional 24-48 hours following infection and then were grown in selective medium containing G418 (800 ug/ml) for 10-12 days prior to staining with
• Producer clones were identified which generated between 5 x 10 4 and 5 x 10 5 G418 resistant colony-forming units per ml.
• X-gal 4-Cl-5-Br-3indolyl- ⁇ -galactoside
• pG1BgSvCb a vector using the bacterial
• ⁇ -galactosidase gene as a selectable markeer and also containing a marked truncated CD4 gene under
• the second is called pG1BgSvCd, a vector similar to pG1BgSvCb but encoding instead the native soluble CD4.
• the first step in the generation of these vectors is the insertion of the 3.0 kb Ncol/Xhol fragment containing the lacZ gene obtained from pBg (see above) and inserting it by blunt ligation into the SnaBI site of pG1, thereby generating pG1Bg.
• the pBg plasmid is also used to construct an SV40 promoted, truncated CD4 gene.
• the construction is begun with the 534 base pair Haell/Nhel fragment of the CD4 gene encoding the amino terminal 178 amino acids of the CD4 receptor.
• an Ncol/Haell linker encoding the 23 amino acid leader sequence of CD4, including the authentic ATG start codon.
• To the 3' end is ligated, in one case, a 45 base pair Nhel/Xhol linker encoding for the bungarotoxin binding domain of the acetylcholine receptor (Btx). This binding domain provides a very sensitive radio-assay for the
• the resulting 601 base pair Ncol/Xhol CD4/Btx fragment is inserted into pBg into the place of the Ncol/Xhol fragment of the lacZ gene to result in the plasmid pCb.
• This 586 base pair Ncol/Hindlll natural CD4 fragment was also inserted into pBg into the place of the
• CD4/Btx gene or the similar Sall/Hindlll fragment of pSvCd were individually ligated to the large
• pGlN2Sv12 The cloning strategy for three other vectors, pGlN2Sv12, pG1N2Sv111, and pG1N2SvI11 provide further examples of the utility of the pGene/pG1 system. All of these vectors may be easily derived directly from pGlN2SvBg, described above, with the gene for ⁇ -gal replaced by one for interleukin-2 (IL-2),
• IL-2 interleukin-2
• interleukin-1 ⁇ IL-1 ⁇
• TNF ⁇ tumor necrosis factor- ⁇
• the IL-2 gene is derived from the plasmid HT-5.1 (ATCC #59396). The 1.0 kb BamHI fragment is isolated from this plasmid and then truncated down to a 445 base pair HgiAI/Dral
• a 100 base pair linker is constructed including the entire 20 amino acid coding region of the amino-terminal end of IL-2, and then a 40 base pair stretch identical in sequence to that of pGene between the Bglll and Ncol sites is added as a 5' leader.
• a SnaBI site is added 5' to the Bglll, permitting direct insertion of this reconstructed IL-2 fragment into pBg which has been digested with SnaBI and Hindlll (the Hindlll blunted with the
• the vector pGlN2SvIll is constructed using a commercially available IL- 1 ⁇ gene obtained from Beckman (catalogue number 267408). The gene is isolated as a 499 base pair NcoI/EcoRI fragment and inserted in the place of the lacZ gene in Ncol/EcoRI digested pBG. An 87 base pair oligomer containing the rat growth hormone secretion signal is then inserted into the Ncol site. The resulting gene can then be removed as a 586 base pair Bglll/BamHI fragment, filled in with Klenow polymerase, and inserted into pGlN2SvBg. The lacZ gene of pGlN2SvBG is removed by digestion with Bglll and Xhol, followed by filling in with Klenow polymerase. These few, simple steps thus yield the final pG1N2SvIll final vector.
• pGlN2SvT11 is constructed similarly.
• the TNF gene is isolated as a 521 NcoI/EcoRI fragment, inserted in the place of the lacZ gene in Ncol/EcoRI digested pBG and has added the identical rat growth hormone secretion signal described above.
• the resulting gene is then removed as a 608 base pair Bglll/BamHI fragment, and inserted into pGlN2SvBg digested with Bglll and Xhol as described above.
• fragments of plasmids may rapidly be exchanged and new vectors can be constructed. If regulation by a different promoter is desirable, a variety of strategies would be available. In the instance of pSvCb, the SV40 promoter could be removed and
• CD4/Btx gene could be removed from pCb and put in the place of a gene running off a
• pG2 a derivative of pG1 called pG2 was constructed.
• a construction strategy for pG2 is shown in Figure 6.
• the difference between pG1 and pG2 is a series of sequence alterations in the 3' LTR which eliminate all enhancer and promoter function without altering the overall structure of the LTR.
• viral replication duplicates the U3 and R portions of the 3' LTR to both the 5' and 3' ends of the proviral integrant.
• the U3 portion of the LTR is 449 base pairs in length and incorporates several regions of strong enhancer activity.
• the R portion of the LTR is 70 base pairs in length and contains the signal for polyadenylation of transcribed mRNAs.
• the R portion is a region of strong
• Clal/Smal fragment was reconstructed with a series of twelve overlapping oligonucleotide fragments. These fragments maintained the overall length of the original LTR, but incorporated sequence alterations which eliminated the recognition sequences for enhancers or promoters. The overall result of this reconstruction was to generate a new Clal/Smal fragment equal in length and general structure to the native fragment, but with all enhancers, distal promoter and TATA regions altered to
• MoMuSV LTR as used to make pG1 was inserted into the unique EcoRI site of the altered pGO to yield the vector backbone pG2. This vector backbone is
• This vector combines all the cloning advantages of pG1, in that the multiple cloning site is
• the pG2 vector provides a useful backbone for the

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