WO2000056874A1 - Vecteurs retroviraux, leurs procedes de production et leur utilisation - Google Patents

Vecteurs retroviraux, leurs procedes de production et leur utilisation Download PDF

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WO2000056874A1
WO2000056874A1 PCT/US2000/007841 US0007841W WO0056874A1 WO 2000056874 A1 WO2000056874 A1 WO 2000056874A1 US 0007841 W US0007841 W US 0007841W WO 0056874 A1 WO0056874 A1 WO 0056874A1
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gene
cell
splice
nucleic acid
retrovirus
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PCT/US2000/007841
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Nancy Hopkins
Wenbiao Chen
Shawn Burgess
Adam Amsterdam
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Massachusetts Institute Of Technology
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/10041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/10043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the invention relates to recombinant retroviral vectors, and the production and uses thereof.
  • candidate genes relies on prior knowledge of the expression pattern or function of a gene and a correlation between these attributes and the mutant phenotype. This approach, by definition, is strongly biased against isolation of genes that have no highly-conserved orthologue. Moreover, it is likely that one could screen tens of candidate gene without finding the mutated gene.
  • a gene trap construct harbors a nucleic acid sequence, such as a reporter gene, that is expressed only when the virus integrates into an active gene.
  • the nucleic acid sequence contains a splice acceptor at the 5' end, allowing for transcription and translation of both the gene that received the insertion and the inserted nucleic acid sequence itself.
  • Introduction of a mutation (a stop codon or a frameshift) after the reporter gene results in truncation (and possibly loss of function) of the interrupted protein.
  • the invention features a recombinant retrovirus including: (a) branch-point sequence; (b) a polypyrimidine tract; (c) a splice acceptor; (d) a splice donor; and (e) LTRs.
  • the splice acceptor and the splice donor flank nucleic acid sequence encoding a stop codon that is in frame with the splice acceptor.
  • the retrovirus includes a reporter gene such as gfp, lacZ, or a nucleic acid encoding myc epitope, a FLAG epitope, or a HA epitope.
  • the reporter gene is preferably in the direction opposite to the direction of transcription from the viral long-terminal repeats. In one preferred embodiment, there are reporter genes in all three reading frames.
  • the retrovirus includes a splice enhancer (e.g., a splice enhancer from the avian sarcoma leukosis virus) or exonic sequence between the splice acceptor and the splice donor.
  • a splice enhancer e.g., a splice enhancer from the avian sarcoma leukosis virus
  • the retrovirus includes nucleic acid sequence encoding a polypeptide encoded in the direction opposite to the direction of transcription from the viral long- terminal repeats.
  • exemplary polypeptides include, but are not limited to, GFP, ⁇ -galactosidase, a myc epitope, a FLAG epitope, a HA epitope, Cre recombinase, and FLP recombinase.
  • the invention features a method for performing gene-trapping in a cell, the method includes (a) contacting the cell with a recombinant retrovirus that includes (i) branch-point sequence; (ii) a polypyrimidine tract; (iii) a splice acceptor; (iv) a splice donor; (v) viral long- terminal repeats; and (vi) a reporter gene in an orientation opposite to the direction of transcription from the viral long-terminal repeats; and (b) allowing the retrovirus to integrate into the genome of the cell.
  • the reporter gene is expressed if there is a gene-trapping event.
  • the cell may be in vitro or in vivo.
  • the invention features a method for introducing a mutation into a gene in a cell, including (a) contacting the cell with a recombinant retrovirus including: (i) branch-point sequence; (ii) a polypyrimidine tract; (iii) a splice acceptor; (iv) a splice donor; and (v) viral long-terminal repeats, wherein the splice acceptor and the splice donor flank nucleic acid sequence encoding a stop codon that is in frame with the splice acceptor; and (b) allowing the retrovirus to integrate into a gene of the cell. Integration of the retrovirus into the gene introduces a mutation into the gene.
  • the method may also include (c) determining the site of integration of the retrovirus.
  • the cell may be in vitro or in vivo.
  • the invention features a method for determining the expression pattern of a gene in a non-human animal, including (a) introducing into the animal or an ancestor thereof a recombinant retrovirus including (i) branch-point sequence; (ii) a polypyrimidine tract; (iii) a splice acceptor; (iv) a splice donor; (v) viral long-terminal repeats; and (vi) nucleic acid sequence between the splice acceptor and the splice donor, the nucleic acid sequence encoding a polypeptide in the direction opposite to the direction of transcription from the viral long-terminal repeats; (b) allowing the retrovirus to integrate into a gene of the animal or the ancestor thereof; and (c) determining the expression pattern of the nucleic acid sequence in the animal, wherein the expression pattern of the nucleic acid sequence mimics the expression pattern of the gene.
  • the animal may be, for example, a mouse, zebrafish, pufferfish, medaka, frog, fly (e.g., a fruit fly), goat, sheep, cow, pig, or chicken.
  • the nucleic acid may include a reporter gene.
  • the invention features a method for producing a transgenic non-human animal, this method includes (a) introducing into an ancestor of the animal a recombinant retrovirus that includes (i) branch-point sequence; (ii) a polypyrimidine tract; (iii) a splice acceptor; (iv) a splice donor; (v) viral long-terminal repeats; and (vi) nucleic acid sequence between the splice acceptor and the splice donor, the nucleic acid sequence encoding a polypeptide in the direction opposite to the direction of transcription from the viral long-terminal repeats; and (b) allowing the retrovirus to integrate into the genome of the ancestor thereof.
  • the animal may be, for example, a mouse, zebrafish, pufferfish, medaka, frog, fly (e.g., a fruit fly), goat, sheep, cow, pig, or chicken.
  • the invention features a method for introducing a nucleic acid sequence into a cell.
  • This method includes contacting the cell with a recombinant retrovirus including (i) branch-point sequence; (ii) a polypyrimidine tract; (iii) a splice acceptor; (iv) a splice donor; (v) viral long- terminal repeats; and (vi) the nucleic acid sequence, and allowing the retrovirus to infect the cell.
  • the cell may be in vitro or in vivo.
  • the invention features a method for identifying a high-titer virus producer cell line, including determining by quantitative PCR the ratio of viral DNA to a control DNA in the cell line.
  • the control DNA is a single copy gene.
  • the invention features a high-titer virus producer cell line identified by determining by quantitative PCR the ratio of viral DNA to a control DNA in the cell line.
  • the invention features a virus produced by the cell line of the eighth aspect.
  • the invention features a method for performing gene therapy on a mammal (e.g., a human), including administering the virus of the eighth aspect to the mammal.
  • a mammal e.g., a human
  • the invention features a method for determining the level of recombinant retroviral infection in a sample from an animal, including determining by real-time quantitative PCR the ratio of viral DNA to a control DNA in the sample.
  • the animal may be, for example, a human or a non-human (e.g., a mouse, zebrafish, pufferfish, medaka, frog, fly, goat, sheep, cow, pig, or chicken).
  • the animal is a human who has undergone or is undergoing gene therapy, and the method is to monitor the efficacy of treatment.
  • branch point sequence is meant a consensus nucleic acid sequence that is recognized in the circularization step in the mRNA splicing reaction (the step in which the 5' end of the intron forms a bond with an adenosine approximately 25 nucleotides upstream of the 3' splice site).
  • polypyrimidine tract is meant a stretch of about 15 nucleotides that are all either cytosine or uracil (cytosine or thymidine in the DNA vector) and that lie between the branch point and the 3' splice site.
  • splice acceptor is meant the nucleic acid sequence at the 3' splice site; this can be used to refer to merely the minimal requirements (an AG dinucleotide or an AG dinucleotide in the context of a short consensus sequence), or more generally to refer to a longer sequence encompassing 30-40 nucleotides upstream of this.
  • a splice acceptor can also include a branch point sequence and a polypyrimidine tract.
  • splice donor is meant the sequence of a 5' splice site; this may refer to either a short consensus sequence, generally beginning with AGGU (AGGT in the DNA vector), or a longer sequence, including specific sequences upstream of the AGGU, that might increase the efficiency of the use of this splice site.
  • LTR long-terminal repeat of a retrovirus, a repeated sequence at both ends of the retrovirus that is required for many steps in the life cycle of the virus, including production of the viral RNA genome in the virus producing cells, proper reverse transcription to create the double stranded DNA provirus in the infected cell, and integration of the provirus into the infected cell's chromosome.
  • the sequence of the 3' LTR in the producer virus- producing cell determines the sequence of both LTRs in the final integrated provirus.
  • reporter gene any gene which encodes a product whose expression is detectable and/or quantitatable by immunological, chemical, biochemical, biological, or mechanical assays.
  • a reporter gene product may, for example, encode a protein having one of the following attributes, without restriction: fluorescence (e.g., gfp), enzymatic activity (e.g., lacZ/ ⁇ -galactosidase, luciferase, chloramphenicol acetyltransferase), toxicity (e.g., ricin), or an ability to be specifically bound by a second molecule (e.g., a FLAG epitope, a myc epitope, a HA epitope, biotin, or a detectably-labelled antibody).
  • fluorescence e.g., gfp
  • enzymatic activity e.g., lacZ/ ⁇ -galactosidase, luciferase, chloramphenicol acetyltransferase
  • toxicity e.g., ricin
  • a second molecule e.g., a FLAG epitope, a myc epi
  • splice enhancer is meant a sequence that resides upstream of a splice donor, and that increases the efficiency of the use of that splice site.
  • exonic sequence is meant sequences that remain in the mRNA following the splicing reaction (i.e., the sequences in the mature mRNA).
  • gene trap is meant a vector containing a nucleic acid sequence gene that can only be expressed when it has inserted into a gene.
  • the nucleic acid sequence might lack a promoter, such that it must integrate downstream of the promoter of an endogenous gene in order to be expressed (often referred to more specifically as a "promoter trap”).
  • promoter trap may lack a promoter, but contain a splice donor, such that if it integrates into an intron of an expressed gene, it will be spliced into the mature RNA of that gene.
  • a gene trap can contain a promoter, but lack other regulatory elements required for efficient gene expression (and thus detection of the reported gene).
  • it can contain a weak promoter which is only activated when integrated near an enhancer (often referred to as an "enhancer trap"), or contain a strong promoter but lack a polyadenylation signal, thus requiring integration upstream of a functional polyadenylation signal for proper expression.
  • an enhancer often referred to as an "enhancer trap”
  • Quantitative PCR is meant a use of the polymerase chain reaction under conditions which can reflect the amount of target sequence (the sequence which is recognized and amplified) in the starting material. This can be achieved, for example, in real-time with the TaqMan system using a
  • recombinase an enzyme which catalyzes DNA recombination reactions, usually in a sequence-dependent manner. This includes the site-specific recombinases from bacteriophage PI (Cre) and yeast (FLP and HO).
  • polypeptide any chain of amino acids, regardless of length or post-translational modification (for example, glycosylation or phosphorylation).
  • high-titer virus producer cell line is meant a cell line, isolated from a parent cell line, that has a viral DNA: single copy gene ratio that is among the top 50% of ratios from cell lines derived the parent cell line.
  • the cell line is in the top 33%, more preferably the cell line is in the top 25%; and most preferably the cell line is in the top 10%.
  • the invention features new gene trap cassettes for retroviral vectors. These cassettes are designed to increase splicing efficiency and to increase viral titer. As a result, the invention provides improved methods for introducing or mutating genes by retroviral-mediated gene trap techniques. The cassette and the methods for its use have a broad range of uses and compatible animal hosts. The invention also provides an improved method for determining viral DNA content in a sample. The method is applicable for identifying high- titer virus producer cell lines (for the production of recombinant viruses for applications that are titer-dependent, such as gene therapy and other types of gene delivery), as well as for quantifying viral DNA in a sample for diagnostic purposes.
  • Fig. 1 shows a schematic illustration of a recombinant retroviral vector containing the GT cassette.
  • the viral LTRs are operably linked to lacZ that has a nuclear localization signal (nlacZ). The direction of transcription from the LTRs is left to right.
  • the GT cassette is in the opposite orientation.
  • SA splice acceptor
  • SD splice donor
  • Fig. 2A shows a schematic illustration of a GT cassette that contains branch point sequence (BPS); a polypyrimidine tract ((Py)n); a splice acceptor (SA); exonic sequence; a splice enhancer; and a splice donor.
  • BPS branch point sequence
  • (Py)n) polypyrimidine tract
  • SA splice acceptor
  • exonic sequence a splice enhancer
  • a splice donor The cassette is designed to result in truncation of the interrupted gene by the introduction of a stop codon or a frameshift in the transcript.
  • Fig. 2B shows a schematic illustration of a GT cassette that is identical to that shown in Fig. 2A, except that it also contains a reporter gene.
  • the reporter gene can be followed by a stop signal.
  • the translation can continue from the reporter sequence into the interrupted gene sequence. In the latter case, translation of the interrupted gene may produce a functional protein.
  • Fig. 2C shows a schematic illustration of a GT cassette that is identical to that shown in Fig. 2B, except that the reporter gene has been replaced by nucleic acid sequence encoding bacteriophage PI Cre recombinase.
  • Fig. 3 shows a schematic illustration of quantitative real-time PCR.
  • oligonucleotide probe nonextendable at the 3' end, labeled at the 5' end, and designed to hybridize within the target sequence, is introduced into the PCR assay. Annealing of the probe to one of the PCR product strands during the course of amplification generates a substrate suitable for exonuclease activity. During amplification, the 5 '-43' exonuclease activity of a suitable DNA polymerase degrades the probe into smaller fragments that can be differentiated from undifferentiated probe. Measurement can be made, for example, on an ABI PRISMTM 7700 sequence detection system
  • Fig. 4 is a graph showing of the range of PCR titering. The results are linear over a broad range of virus concentration.
  • Fig. 5 shows a schematic illustration of the steps for isolation of high-titer virus producer cell lines.
  • Fig. 6 is a graph showing the correlation between PCR titer and recombinant retroviral infection in zebrafish embryos.
  • Insertional mutagenesis allows for rapid cloning of the mutated genes. So far we have cloned candidate genes for six of the seven insertional mutants. Although, with present techniques, it is theoretically possible to generate enough insertions in the fish germ line to mutate all the genes, it is laborious to breed the fish harboring insertions to homozygosity in order to determine which have induced (i.e., recessive) embryonic mutations. In addition, the frequency of mutagenesis with the available viruses is rather low and integration events that occurred in large introns may not perturb gene expression and, thus, may not always appear mutagenic.
  • viruses described herein also contain a novel gene-trapping module (named GT). The viruses that include this gene-trapping module differ from existing gene-trapping viruses in several regards. The general improvements are shown in Fig. 1 and are described below.
  • the viruses of the present invention have all of the elements necessary for efficient RNA splicing, including branch-point sequence, polypyrimidine tract, and a splice acceptor and splice donor flanking a mini-exon (see below). The presence of these elements facilitates recognition of and splicing with the inserted ex on with the trapped gene.
  • Gene-trapping viruses normally have a terminal exon encoding a reporter gene followed by polyadenylation sequence, thus leading to a truncation of the mRNA and utilization of a polyadenylation sequence that is not endogenous to the gene that has been trapped.
  • viruses described herein have a mini-exon between the splice acceptor and splice donor. Transcription continues from an endogenous exon, through the mini- exon, and to the next exon of the trapped gene. Moreover, polyadenylation is via the endogenous polyadenylation sequence. The utilization of the endogenous polyadenylation sequence is likely to increase the amount of mRNA that is produced.
  • the artificial mini-exon also harbors a pyrimidine-rich splice enhancer from avian sarcoma leukosis virus (ASLV) to augment its recognition by cellular RNA splicing machinery.
  • ASLV avian sarcoma leukosis virus
  • the mini-exon encodes a small peptide epitope, such the FLAG epitope (DYKDDDDK) in one, two, or all three reading frames.
  • the provirus integrates in an intron in the correct orientation, the artificial exon is spliced into the mRNA.
  • the mini-exon can be designed so that it causes a frameshift mutation.
  • An additional advantage of the gene trap cassette described herein is its small size, which contributes to the virus having a high titer.
  • the increased titer allows for more integration events per animal, which is important in the generation of mutants.
  • Cloning of the gene into which the virus integrates can be performed by RACE (e.g., 3' RACE or 5' RACE (Rapid Amplification of cDNA Ends)).
  • RACE e.g., 3' RACE or 5' RACE (Rapid Amplification of cDNA Ends)
  • the occurrence of an integration event can also be detected by RT-PCR, in situ RNA hybridization, or immunodetection using antibodies against a peptide epitope.
  • RNA from unfertilized eggs of female founders we PCR-amplified the cDNAs with a second mini-exon-specific primer (primer 2) internal (i.e., 5') to primer 1 and a 5' RACE abridged anchor primer.
  • a nested PCR was then performed on the PCR products using a third mini-exon-specific primer (primer 3) and an abridged universal amplification primer.
  • the PCR products were gel-purified and sequenced to determine whether the amplified products were of authentic hybrids of the mini-exon and mRNA.
  • Three of the 15 samples have fragments from two viral integration events, as determined by sequence analysis. Thus, there is, on average, at least one detectable gene-trap event per founder. Similar conclusions were reached by analyzing RNA from unfertilized eggs of female founders.
  • the viruses described above have many uses, including gene trap- mediated mutagenesis, gene expression analysis, and gene delivery.
  • the viruses can encode a site-specific recombinase, allowing for conditional mutation of a gene that has sites recognized by one of the foregoing recombinases.
  • the viral vector can itself include loxP or FRT sites, such that the reporter gene or the entire viral vector can be removed from the host genome. Each of these uses is discussed in detail below. Mutagenesis
  • the viral vectors containing the GT cassette are useful for gene trap- mediated insertional mutagenesis.
  • Animals containing one or more proviral insertions are produced using standard techniques known to those skilled in the art (e.g., Canrey et al., Dev. Dyn. 212:284-292, 1998).
  • the GT cassette includes, between the splice acceptor and splice donor, nucleic acid sequence encoding a stop codon in one, two, or all three reading frames (Fig. 2A).
  • the mini-exon itself can, by virtue of its nucleic acid length not being a multiple of three, lead to a frameshift that should result in a premature stop or non-functional protein.
  • integration of the provirus may lead to a gene mutation and a resulting gross morphological or physiological phenotype.
  • Determination of the expression pattern of the interrupted gene can also be performed using a viral vectors containing a GT cassette (Fig. 2B).
  • the mini-exon contains nucleic acid sequence encoding a reporter polypeptide, such as a peptide epitope (e.g., FLAG, HA, or myc). It is preferred if all three reading frames encode a reporter polypeptide, as this assures the proper translation of a reporter polypeptide. Moreover, as we have found that smaller GT cassettes result in higher viral titer, small reporter polypeptides (such as the foregoing peptide epitopes) are preferred. Expression of the reporter polypeptide will be under control of the promoter of the interrupted gene.
  • a reporter polypeptide such as a peptide epitope (e.g., FLAG, HA, or myc). It is preferred if all three reading frames encode a reporter polypeptide, as this assures the proper translation of a reporter polypeptide.
  • small reporter polypeptides such as the foregoing peptide epitopes
  • Expression of the reporter polypeptide will be under control of the promoter of the interrupted gene.
  • detection such as immunodetection of a peptide epitope
  • detection of the expression pattern of the reporter polypeptide will reveal the expression pattern of the interrupted gene.
  • Other methods of detection include, but are not limited to, RT-PCR, in situ hybridization, western blot analysis, and detection of enzymatic activity or fluorescence.
  • GT cassette-containing retroviruses described herein Another use of the GT cassette-containing retroviruses described herein is to express a nucleic acid of interest in a spatio-temporally dynamic manner.
  • the disadvantages to this approach in comparison to expressing a gene of interest from a defined promoter element, are that one cannot predict from the outset the resulting expression pattern, or if the interruption of a gene will result in a loss of function of that gene.
  • the advantages include the relative ease of producing large numbers of lines of animals, and the ability to achieve an expression pattern not previously identified with a known promoter.
  • the method is, in essence, identical to that described in the previous paragraph, except that the nucleic acid encoding the reporter polypeptide is replaced with the nucleic acid of interest.
  • the expression pattern can be determined using standard techniques such as, for example, RT-PCR, in situ hybridization, immunohistochemistry, and western blot analysis.
  • One particular set of preferred nucleic acids those encoding site- specific recombinases, such as the bacteriophage PI Cre recombinase or FLP recombinase that have enzymatic activity when expressed in a vertebrate (Buchholz et al., Nuc. Acids Res. 24:4256-4262, 1996) .
  • site- specific recombinases such as the bacteriophage PI Cre recombinase or FLP recombinase that have enzymatic activity when expressed in a vertebrate (Buchholz et al., Nuc. Acids Res. 24:4256-4262, 1996) .
  • Each of these recombinases recognizes a sequence motif, loxP and FRT, respectively; DNA flanked by two such sequence motifs is excised by the recombinase.
  • strategies have been developed in which conditional expression of a gene is regulated by recombinase
  • a mouse having loxP sites, engineered into introns flanking exon 2 of gene X can be mated with a second mouse expressing Cre recombinase only in the central nervous system.
  • cells in the central nervous system will have Cre recombinase activity, and exon 2 of gene X will be excised. In all other tissues, exon 2 will not be excised.
  • lines of animals can be produced, each of which having a unique expression pattern of recombinase (Fig. 2C). These animals can be mated to mice having loxP or FRT sites flanking a gene or gene segment, resulting in numerous lines of animals, each having a different pattern of gene inactivation.
  • the viruses of the invention can also have loxP or FRT sites themselves (Russ et al., J. Virol. 70:4927-4932).
  • placement of loxP sites in the 5' and 3' LTRs allows for reversible gene interruption.
  • the provirus has inserted into gene Y of a mouse, resulting in a truncation of the encoded protein and loss of function.
  • the mutant mouse is then mated with a second mouse, in which Cre recombinase is expressed throughout the animal.
  • Cre recombinase will excise nearly the entire proviral sequence, resulting in restoration of protein function.
  • the methods and viruses described herein have uses in a wide variety of animals that can be infected with retroviruses, including animals used in scientific research (e.g., mice, zebrafish, pufferfish, medaka, frog, and fly), and those with commercial value (e.g., goats, sheep, cows, pigs, and chickens).
  • the GT cassette can be readily adapted to any retroviral vector, and, if required, the VSV-G viral envelope can be employed for infection of nearly any vertebrate cell.
  • a producer cell line is usually selected from a pool of candidates. Traditionally, it involves serial dilution of conditioned medium from individual candidate clones. These dilutions are then used to infect indicator cells (e.g. NIH 3T3), and the number of infection events is determined by counting the clones that express a viral marker (e.g., a visible marker such as lacZ or a selectable marker such as neo).
  • a viral marker e.g., a visible marker such as lacZ or a selectable marker such as neo.
  • This approach takes more than a week from time of infection to colony counting, and as a result requires intensive tissue culture manipulations to maintain the candidates in the duration. Consequently, usually only a few dozen candidate clones are usually tested. While this might result in a high-titer producing clone, it is statistically more likely that a larger initial pool of candidates will result in a better clone.
  • a high throughput screening method to isolate retrovirus producer cell lines. Using this method, a high-titer GT virus producer cell line was quickly selected from 230 candidates.
  • This method uses real-time quantitative PCR analysis to compare the number of proviral insertions in target cells transduced by the conditioned medium of individual candidate.
  • the PCR assay uses ABI PRISMTM 7700 sequence detection system (PE Applied Biosystems, Foster City, CA) to quantify PCR product accumulation through a dual-labeled virus specific fluorogenic probe (i.e., TaqMan Probe). Briefly, an oligonucleotide probe, nonextendable at the 3' end, labeled at the 5' end, and designed to hybridize within the target sequence, is introduced into the PCR assay.
  • Annealing of the probe to one of the PCR product strands during the course of amplification generates a substrate suitable for exonuclease activity.
  • a DNA polymerase e.g., Taq polymerase
  • Fig. 3 Holland et al., Proc. Acad. Natl. Sci. USA 88:7276-7280, 1991 ; Heid et al. Genome Res. 6:986-994, 1996.
  • Quantitative data are derived from a determination of the cycle at which the fluorescence reaches a preset detection threshold. The earlier the threshold is reached, the more target in the sample.
  • the assay is very accurate and reproducible (Fig. 4). The selection procedure is outlined below.
  • Candidate clones were made by infecting 293bsr, a packaging cell line, with GT virus followed by fluorescent activated cell sorting (FACS) of infected cells.
  • GT virus was prepared by transient co-transfection of pCMV-GT and pCMV- VSV-G. FACS was performed two days post-infection after loading infected cells with FDG, a membrane-permeable fluorogenic substrate of ⁇ -galactosidase. Cells with the strongest fluorescence (top 0.5%) were collected and seeded individually into 96- well plates (Fig. 5). Once grown to confluence, cells in each well were transferred to a compartment in 24-well plates.
  • cells were divided into six parts and each part seeded in a well of one of six 96-well plates. The remaining cells were maintained in the same compartment. The next day, the cells were transfected with pCMV- VSV-G (by calcium phosphate co-precipitation or lipofectamine), then cultured in lOO ⁇ L of medium.
  • pCMV- VSV-G by calcium phosphate co-precipitation or lipofectamine
  • VSV-G coat viruses can infect a broad range of hosts, the in vitro titering of virus should be performed on cells of the same animal type will be infected in vivo.
  • 25 ⁇ L of the medium in each well was taken to infect PAC2 cells (a zebrafish cell line) in 96 well plates.
  • the PAC2 cells were lysed in 25 ⁇ L GNT buffer. Lysate (2 ⁇ L) from each well was analyzed by simultaneous real-time quantitative PCR analysis for viral DNA and an endogenous single-copy gene, RAGl .
  • the ratio of viral DNA to RAGl DNA were then determined, and the top 10% in each transfection method of the first 230 clones were kept for second round of selection. After repeating the above procedure, the top six clones from each transfection method were chosen for large-scale preparation.
  • the titer of each of the clones was determined by quantitative PCR, and quantitative Southern analysis in injected fish larvae. As predicted, there is a very strong correlation between the two assays (Fig. 6).
  • Clone 186 was selected from 230 candidates to make GT virus by lipofection. Because lipofection is a more robust method of transfection, it was chosen for subsequent studies. To optimize lipofection conditions, we compared the titer of virus made by the combination of 5, 10, 15, and 20 ⁇ g of pCMV- VSV-G DNA per 15 cm plate at ratios of DNA to lipofectamine of 1: 10, 1 : 14, 1 :20, and 1:30 (w/w). Of those, 5 ⁇ g DNA, 75 ⁇ l lipofectamine per plate produced virus with high titer and low toxicity.
  • clone 186 produced viruses that generate, in injected fish, an average of 20 proviral insertions per cell after one round of injection, compared to 4.5 after two rounds of injection using the existing control cell line.
  • the survival rate following injection also increased from 40% to more than 65%.
  • the combination of these factors has increased the weekly output of adult founders from 600 to more than 3000.
  • This rapid and high throughput assay for high-producer cells has other advantages over conventional screening procedure. First, it does not require a marker gene in the virus. Most retroviral vectors, in particular those for gene therapy purposes, contain a selectable marker gene specifically for selecting a producer cell line.
  • Inclusion of the extra sequence may not only lower the titer by promoter suppression, but also increase the immunogenicity of host cells. It is desirable to make viral vectors that do not have a selectable marker.
  • the method described herein is useful for selecting good producer cells for these very viruses. Second, the method selects clones based on the titer on cells derived from the target species. Third, the method described herein is more reliable than methods that measure viral RNA content in conditioned medium. Viral RNA is a poor predictor of infection, especially for cells from non-mammals such as zebrafish. Finally, it is much less labor- intensive than methods that uses competitive quantitative PCR analysis.
  • Himiaii gene therapy Production of sufficient amounts of high-titer virus to infect large numbers of specific cell populations for the treatment of human diseases has been a challenging problem.
  • the technology described here allows one to rapidly identify cell clones, harboring the viral vector of choice, that produce high titers of virus that are capable of infecting the cell of interest.
  • Insertional mutagenesis makes it possible to rapidly clone genes required for any biological process of interest.
  • To isolate mutations in the genes of interest requires that one generate a large number of proviral insertions in the germ line of the species of interest. Animals harboring the insertions are then bred so as to bring the insertions to homozygosity and mutant animals are then identified by screening for the phenotype(s) of interest.
  • the method described herein allows one to isolate clones of cells producing very high-titer stocks of virus for infecting virtually any animal species.
  • the technology opens the possibility of using retroviral vectors to perform insertional mutagenesis in a wide variety of animal species.
  • the cell lines identified by the method described herein provide a means for producing high-titer recombinant virus containing large inserts. This allows for gene mis-expression, production of transgenic animals, or gene therapy in cases where the viral titer produced using previous means was inadequate.
  • the method for quantifying virus described herein has other uses.
  • Cells such as stem cells, that might be infected before being transplanted into an animal, can be quicky assayed for viral DNA content.
  • diagnostic assays for infection levels can also use this technique.
  • quantifying recombinant retroviral DNA is useful in determining whether a batch of injected animals are likely to have a high frequency of proviral insertions. In generating insertional mutants, we have observed a strong correlation among animals injected with a given viral preparation by a given person.
  • the particular cell clone described here which produces very high- titer virus stocks for infecting the zebrafish germ line, contains a viral vector that includes a gene trap cassette. This cassette will likely lead to a large increase in the mutagenic frequency of the virus and will accelerate the identification of mutants and the subsequent cloning of mutated genes.
  • the expression of genes in transgenic animals can be valuable for basic research purposes and in some cases for commercial purposes directly.
  • Vectors would be expected to express genes in a wide variety of fish as well as in other animals, such as chickens, cows, pigs, and sheep.
  • the methods described herein are useful for generating cell clones producing high titers of any retroviral vector construct.

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Abstract

L'invention concerne des procédés et des réactifs servant à introduire une mutation dans un gène d'une cellule. Ces procédés consistent à mettre en contact la cellule avec un rétrovirus recombiné comprenant (I) une séquence de point de bifurcation; (ii) une partie polypyrimidine; (iii) un accepteur d'épissage; (iv) un donneur d'épissage; et (b) une longue répétition terminale virale, l'accepteur et le donneur d'épissage flanquant une séquence d'acide nucléique qui code un codon d'arrêt situé à l'intérieur d'un cadre avec l'accepteur d'épissage. Ces procédés consistent également à permettre l'intégration du rétrovirus dans un gène de la cellule, cette opération entraînant une mutation dans le gène.
PCT/US2000/007841 1999-03-25 2000-03-24 Vecteurs retroviraux, leurs procedes de production et leur utilisation WO2000056874A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6897020B2 (en) 2000-03-20 2005-05-24 Newlink Genetics Inc. Methods and compositions for elucidating relative protein expression levels in cells
EP1268767B1 (fr) * 2000-03-20 2005-12-07 Newlink Genetics Procedes et compositions servant a identifier des profils d'expression de proteines dans des cellules
US7625755B2 (en) * 2003-05-30 2009-12-01 Wyeth Conditional knockout method for gene trapping and gene targeting using an inducible gene silencer

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5679523A (en) * 1995-11-16 1997-10-21 The Board Of Trustees Of The Leland Stanford Junior University Method for concurrent disruption of expression of multiple alleles of mammalian genes

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5679523A (en) * 1995-11-16 1997-10-21 The Board Of Trustees Of The Leland Stanford Junior University Method for concurrent disruption of expression of multiple alleles of mammalian genes

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BOUCK, J. ET. AL.: "In vivo selection for intronic splicing signals from a randomized pool", NUCLEIC ACIDS RESEARCH, vol. 26, no. 19, 1998, pages 4516 - 4523, XP002929462 *
BOUCK, J.: "Role of the Constitutive Splicing Factors U2AF65 and SAP49 in Suboptimal RNA Splicing of Novel Retroviral Mutants", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 273, no. 24, 12 June 1998 (1998-06-12), pages 15169 - 15176, XP002929461 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6897020B2 (en) 2000-03-20 2005-05-24 Newlink Genetics Inc. Methods and compositions for elucidating relative protein expression levels in cells
EP1268767B1 (fr) * 2000-03-20 2005-12-07 Newlink Genetics Procedes et compositions servant a identifier des profils d'expression de proteines dans des cellules
US7625755B2 (en) * 2003-05-30 2009-12-01 Wyeth Conditional knockout method for gene trapping and gene targeting using an inducible gene silencer

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