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WO2000065076A2 - Gene synthetique destine a l'expression d'une proteine retrovirale active dans les eucariotes - Google Patents

Gene synthetique destine a l'expression d'une proteine retrovirale active dans les eucariotes Download PDF

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WO2000065076A2
WO2000065076A2 PCT/EP2000/003765 EP0003765W WO0065076A2 WO 2000065076 A2 WO2000065076 A2 WO 2000065076A2 EP 0003765 W EP0003765 W EP 0003765W WO 0065076 A2 WO0065076 A2 WO 0065076A2
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gene
protein
retroviral
integrase
synthetic gene
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PCT/EP2000/003765
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WO2000065076A3 (fr
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Zeger Debyser
Erik De Clercq
Peter Cherepanov
Wim Pluymers
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K.U. Leuven Research & Development
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Priority to BR0010077-3A priority Critical patent/BR0010077A/pt
Priority to IL14609000A priority patent/IL146090A0/xx
Priority to CA002369058A priority patent/CA2369058A1/fr
Priority to EP00927069A priority patent/EP1183381A2/fr
Priority to AU45579/00A priority patent/AU778106B2/en
Priority to JP2000614410A priority patent/JP2002542791A/ja
Publication of WO2000065076A2 publication Critical patent/WO2000065076A2/fr
Priority to NO20015168A priority patent/NO20015168L/no
Publication of WO2000065076A3 publication Critical patent/WO2000065076A3/fr

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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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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
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16051—Methods of production or purification of viral material
    • C12N2740/16052—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/108—Plasmid DNA episomal vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES

Definitions

  • the present invention relates to the design of a synthetic gene for expressing retroviral proteins in eukaryotic cells especially mammalian cells as well as a synthetic gene, an expression vector containing the gene, eukaryotic cells stably harboring the gene, as well as methods of detection.
  • Retro viruses are diploid positive strand RNA viruses that replicate through an integrated DNA intermediate.
  • retroviruses comprise a protein-containing lipid envelope surrounding a protein-encapsulated core carrying the viral genome.
  • the retroviral genome is reverse-transcribed into double stranded DNA by a virally encoded reverse transcriptase enzyme that is part of the retroviral particle.
  • the particle also includes other enzymes such as integrase. Integrase is the virus-encoded enzyme that is responsible for inserting the viral DNA copy into the chromosome of the host cell, a process referred to as retroviral integration.
  • Integration is an essential step in the replication cycle of the human immunodeficiency virus type 1 (HIN-1), the causative agent of AIDS (LaFemina et al. (1992), J. Virol. 66: 7414-7419). Since no human counterpart is known to exist, integration has attracted a lot of attention as a potential new antiviral target. However, integrase inhibitor development has suffered from the lack of a relevant cellular integration assay; integrase activity is typically evaluated using artificial oligonucleotide-based test tube reactions. There is therefore a need to provide an intracellular integration assay.
  • HIN-1 human immunodeficiency virus type 1
  • AIDS the causative agent of AIDS
  • Wild-type retroviral genomes contain at least three genes known as the gag, pol and env genes.
  • the gag gene encodes internal core structural proteins
  • the pol gene encodes for certain enzymes such as protease, reverse transcriptase and integrase
  • the env gene encodes the retroviral envelope glycoproteins.
  • Integrases from different retroviruses vary in size from 30 to 46 kDa, are encoded by the 3' -end of the pol gene and are released from a gag-pol polyprotein precursor by proteolytic processing.
  • the aminoterminal domain of integrase is characterized by a zinc finger (HHCC), is universally conserved among all retroviruses, and is essential for in vivo integration.
  • HHCC zinc finger
  • the central domain is the most conserved region with an essential DD35E motif involved in catalysis. This portion can catalyze the disintegration reaction in vitro.
  • the carboxyterminal domain is referred to as DNA binding domain and shows the least sequence conservation. This fragment is required for 3 '-end processing and integration.
  • the active enzyme is thought to exist as a multimer wherein active domains can transcomplement inactive domains.
  • avian sarcoma-leukosis virus (ASLV) integrase in COS cells has been obtained previously (Morris- Vasios et al. (1988), J. Virol. 62: 349-353).
  • a mouse cell line stably expressing the integrase of Rous sarcoma virus (RSV) has also been reported (Mumm et al. (1992), Virology 189: 500-510). Expression levels were not specified but appeared rather low.
  • the integrase (IN) of HIV- 1 has been expressed in Escherichia coli (E. col ⁇ ) (Sherman and Fyfe (1990), Proc. Natl. Acad. Sci.
  • US 5,811,270 (Grandgenett) describes a test tube method of analysis of concerted integration in which a viral integrase enzyme is first incubated with donor DNA molecules followed by incubation with target DNA molecules.
  • the donor DNA has at least one unique restriction site for analysis of the concerted integration product.
  • the described method is said to be useful for studying integrase such as screening of HIV-1 or HIV-2 integrase inhibitors as well as production of transgenic non-human animals and gene transfer.
  • the integrase used is purified from virus particles and the activity is analyzed in the test tube, not intracellularly.
  • a retroviral protein which fails to show intracellular enzymatic activity.
  • the expressed enzyme may be defective for many reasons of which intracellular inhibition of the enzyme and the need for the presence of another viral protein at the same time are but a few. Further, it is not obvious that an enzyme can be overexpressed, for example there may be some limiting factor such as poor solubility or cellular toxicity.
  • high level expression of a retroviral enzyme will be required to detect the enzymatic activity, on the other hand levels which are too high may cause protein precipitation or cellular toxicity. For any retroviral enzyme to be active in the cell an optimal intracellular concentration will be required.
  • a further object of the present invention is to provide an expression vector capable of delivering a gene to a target cell, in which cell the enzymatically active protein encoded by the gene is expressed.
  • the present invention features a synthetic gene or region of a gene which has an amended codon usage compared with the wild-type gene and which is for the high level expression of a retroviral protein in eukaryotic cells, the expressed retroviral protein having enzymatic activity in the eukaryotic cell.
  • the invention features a synthetic gene or region of a gene encoding a retroviral enzyme or part of a retroviral enzyme normally expressed in a mammalian or other eukaryotic cell wherein at least one non-preferred codon in the wild-type gene encoding the enzyme has been replaced by a preferred codon encoding the same amino acid.
  • region of a gene with amended codon usage is meant that it can be sufficient to change codons only in those parts of a gene that normally produce instability sequences (INS) or cis-acting repressor elements (CRS) in the transcribed mRNA of the gene.
  • INS instability sequences
  • CRS cis-acting repressor elements
  • retroviral protein or enzyme normally expressed in a mammalian or eukaryotic cell is meant a protein or enzyme which is expressed in a mammalian or eukaryotic cell under disease conditions. These are genes which are encoded by a retro virus (including a lentivirus) which are expressed in mammalian or eukaryotic cells post-infection.
  • the synthetic gene is capable of expressing the retroviral enzyme at a level at least 200% of that expressed by the "natural" (or "native") gene in a mammalian or eukaryotic cell culture system.
  • the retroviral protein may be a protease, reverse transcriptase, integrase protein or a polyprotein gag-pol precursor thereof.
  • the retroviral protein with enzymatic activity is a lentiviral protein.
  • the enzymatically active protein is a pol enzyme.
  • the enzymatically active protein is a lentiviral integrase.
  • the enzyme is an HIV enzyme.
  • the enzymatically active protein is HIV integrase.
  • the enzymatic activity includes at least an integrase function, namely of promotion or stimulation of the integration of DNA fragments into host cell DNA, preferably the chromosome of the host cell.
  • the integrase hereby is expressed on its own id est as a single component, independent of any retroviral components.
  • retroviral components is meant the retroviral, specifically the lentiviral, and more specificially the HIV-1 regulatory and accessory proteins like Tat, Rev, Nef, Vpu, Vif, Vpr.
  • the invention also features a eukaryotic expression vector comprising the synthetic gene or region of a gene.
  • the expression vector preferably includes a constitutive or an inducible or a tissue-specific promoter. Expression from the eukaryotic expression vector can be transient after transfection of the vector in a eukaryotic cell by any of suitable, e.g. established, transfection procedures.
  • the vector may be any suitable vector such as a plasmid, a mammalian or insect virus. Expression may also be permanent in a eukaryotic cell line stably harbouring the expression vector.
  • the expression vector may be comprised in a packaging construct for producing retroviral particles for gene transfer.
  • the retroviral particle may be a lentiviral particle.
  • Another aspect of the present invention features a eukaryotic cell line that harbours the synthetic gene or region of a gene.
  • the cell line preferably expresses the retroviral enzymatically active protein using a constitutive, inducible or tissue specific promoter.
  • the expressed retroviral protein shows enzymatic activity that can be measured for example by complementation of enzyme-defective viruses or in the case of an integrase by stimulation or the promotion of the insertion of DNA molecules into another DNA molecule, preferably the chromosome of the cell.
  • the present invention also includes a transgenic non-human animal harboring the synthetic gene or region of a gene.
  • the expression of the gene or region of a gene may be induced at any moment using an inducible promoter or, alternatively, in desired tissues using a tissue-specific promoter.
  • the present invention also features a method for preparing a synthetic gene or region of a gene encoding an enzymatically active retroviral protein or part of such a protein.
  • the method not only identifies and uses preferred codon usage but also, and moreover mainly, seeks to increase mRNA stability during expression.
  • the method includes identifying a small group of genes from the total set of genes of a target eukaryotic cell which encode proteins which are naturally expressed easily and/or in high concentrations in the target cell.
  • the small group may include 10 or less genes, more typically 5 or less genes. From the codon sequences of these identified genes, a preferred codon usage and a preferred nucleotide relationship or nucleotide pair frequency is identified.
  • preferred codon usage is meant that for a specific amino acid a specific codon is chosen as the preferred codon to encode the amino acid based on the high use of the preferred codon within the select group of genes.
  • a preferred codon relationship is meant the ratios of the various nucleotides and combinations of nucleotides to each other which commonly appear in genes of the target eukaryotic cell.
  • One particular nucleotide relationship is the GC content or the GC nucleotide pair frequency.
  • non-preferred codons are identified in the natural gene encoding the enzyme and one or more of the non-preferred codons is/are replaced with a preferred codon encoding the same amino acid as the replaced codon.
  • the replacement is biased to obtain the preferred nucleotide relationship or nucleotide pair frequency, resulting in even better optimized conditions for expression in eukaryotes compared to the use of preferred codon usage only.
  • the replacement may be made based on a random choice between alternative codons encoding the same amino acid at each position using a random number generator and biasing the choice of alternative codons based on the preferred codon usage to obtain the preferred nucleotide relationship or nucleotide pair frequency.
  • the synthetic gene sequence may be edited by removing potential splice sites and to reduce the number of CpG methylation sites while keeping the overall nucleotide relationship or the nucleotide pair frequency close to the preferred one, e.g. keeping the GC content and codon usage close to the preferred one.
  • GC content should be kept close to the preferred usage in the target cell, e.g. about 60%> in mammalian cells.
  • a preferred range for the GC content is 53 to 63%, more preferably 55 to 61% for expression of the gene in human cells.
  • the Kozak consensus sequence (ANNATGG) may be added. It is not necessary to replace all non-preferred codons with preferred codons.
  • Gene is meant a nucleotide sequence encoding a naturally occurring protein in which a portion of the naturally occurring codons has been replaced by other codons. For example, a non-preferred codon is replaced with a preferred codon encoding the same amino acid. However, by replacing codons to create a synthetic gene the expression in eukaryotic, e.g.
  • mammalian cells especially human cells
  • genes of eukaryotic, mammalian, prokaryotic or viral origin
  • the invention includes improving the eukaryotic, especially a mammalian cell expression of a gene from any source by the codon replacement methods described herein.
  • vector is meant a DNA molecule, derived, e.g., from a plasmid, or mammalian or insect virus, into which fragments of DNA may be inserted or cloned.
  • a vector will contain one or more unique restriction sites and may be capable of autonomous replication in a defined host or vehicle organism such that the cloned sequence is reproducible.
  • expression vector is meant any autonomous element capable of directing the synthesis of a protein.
  • DNA expression vectors include mammalian plasmids and viruses.
  • retroviral "packaging construct” or “packaging vector” is meant a plasmid- based or virus-based vector or construct, configured to encode for the proteins necessary for producing virus particles that are devoid of genomic RNA. In general, this implies providing the gag, pol and env gene products.
  • Lentiviral packaging constructs of interest contain changes to the coding sequences of gag or pol proteins (i.e. synthetic genes) to enhance lentiviral protein expression and to enhance safety. For biosafety reasons, the packaging functions are often divided into two genomes, one which expresses the gag and pol gene and another expressing the env gene product. In packaging constructs in accordance with the present invention, regulators of gene expression such as the Rev gene product would no longer be required.
  • the invention also features synthetic portion of a gene which encodes a desired portion of the protein.
  • Such synthetic gene fragments are similar to the synthetic genes of the invention except that they encode only a portion of the protein.
  • the portion of the gene encodes a portion of the enzyme which has some enzymatic activity, e.g. it may have catalytic activity, for example, the synthetic gene may encode a catalytic core of an enzyme, e.g. it may be a part of reverse transcriptase.
  • the present invention also includes a detection method for intracellular integrase using a promoterless reporter gene.
  • the reporter gene may be luciferase, GFP or an antibiotic selection marker (e.g. neomycin resistance).
  • the reporter gene construct may be used as the substrate of the retroviral enzyme, e.g. integrase expressed from the synthetic gene be it in a stable cell line or in a transient mode after transfection of the expression vector, the retroviral enzyme, e.g. integrase being in accordance with the present invention.
  • the present invention may provide a synthetic gene and a method of designing and constructing the same to obtain efficient expression of a retroviral, in particular lentiviral enzyme such as integrase of the human immunodeficiency virus type 1 (HIV- 1), or part of a retroviral enzyme in mammalian cells.
  • a retroviral, in particular lentiviral enzyme such as integrase of the human immunodeficiency virus type 1 (HIV- 1)
  • the synthetic gene circumvents mRNA instability by increasing the GC content of the wild type integrase gene from 40% to 59%.
  • the synthetic gene, cloned in a eukaryotic expression vector provides efficient expression of HIV-1 integrase in various mammalian cell lines. The amino terminus of the protein was as predicted by the sequence after removal of the first methionyl residue.
  • FIG. 1 Western blot analysis of transient expression of HIV-1 IN in 293T cells using different expression strategies. 293T cells were transiently transfected with the various expression vectors. At 48 hrs post transfection cell extracts were made using 1% SDS, 1 mM PMSF. Cell extracts representing 10 ⁇ g of total protein were separated by PAGE and blotted onto PVDF membranes. Detection was performed using polyclonal antibodies against HIV-1 integrase and the ECL+ detection system. Lane 1 contains 2.5 ng of recombinant and purified His-tagged HIV-1 integrase (HT- IN).
  • the other lanes contain extracts after transfections with equal amounts of the following plasmids: Lane 2, pCEP4; Lane 3, pCEP-IN; Lane 4, pCEP-IN-CTE; Lane 5, pCEP-IN-RRE + pEF-cREV; Lane 6 pCMV-IN s .
  • FIG. 1 A schematic representation of the structure of the synthetic gene. The following regions are indicated : the 5'- and 3'- untranslated regions (UTR) derived from ⁇ - globin mRNA, the Met-Gly dipeptide and the integrase open reading frame (ORF). The three domains of the integrase protein are shown: the Zinc finger motif (HHCC), the catalytic core and the DNA binding domain.
  • UTR 5'- and 3'- untranslated regions derived from ⁇ - globin mRNA
  • Met-Gly dipeptide the Met-Gly dipeptide
  • ORF integrase open reading frame
  • the three domains of the integrase protein are shown: the Zinc finger motif (HHCC), the catalytic core and the DNA binding domain.
  • FIG. 3 Western blot analysis of the 293T-derived cell line that stably expresses HIV-1 IN from the synthetic gene.
  • 293T cells were transfected with pCMV-IN s and a stable cell line was selected with HygromycinB.
  • Cell extracts (10 ⁇ g of total protein) were separated by PAGE and blotted onto PVDF membrane. Detection was performed using polyclonal antibodies against HIV-1 integrase and the ECL+ detection system. Lane 1, 2.5 ng recombinant His-tagged HIV-1 integrase; Lane 2, extract of 293T cells; Lane 3, extract of 293T cells stably expressing IN (293T-IN S ).
  • Figure 4A-C are schematic representations of the method of detection of integrase activity using a promoterless reporter gene.
  • the present invention will mainly be described with reference to a synthetic gene for overexpressing HIV integrase in mammalian cells but the invention is not limited thereto but only by the claims.
  • a retroviral enzyme such as integrase does not normally, during the infectious cycle, work as a soluble protein in the cytoplasm of a host cell. Integrase is part of a large ill-defined nucleoprotein complex called the preintegration complex of which also reverse transcriptase, nucleocapsid, matrix protein, the viral
  • DNA and other factors are part. It is not obvious that integrase on its own in the cytoplasm of a target cell is enzymatically active, for example, there may be cellular factors which inhibit activity or viral factors which are missing in this environment. Further, it is not obvious that integrase expressed as such will interact with artificial DNA substrates (see DIPR below).
  • One aspect of the present invention is dissecting the preintegration complex to obtain a simple integrase-linear DNA interaction.
  • One embodiment of the present invention is a method to detect and utilize the enzymatic activity of a retroviral, in particular a lentiviral enzyme, in particular integrase by itself in a eukaryotic cell.
  • the impact of the INS in the IN gene on protein expression levels was illustrated by a 5-fold decrease in expression levels of the GFP- IN construct compared to the parental GFP (Pluymers et al. (1999), Virology 258: 327- 332).
  • the present invention is based on a synthetic gene for HIV-1 integrase with an increased intrinsic mRNA stability. The use of such a synthetic gene resulted in high expression levels and concurrent enzymatic integrase activity as could be demonstrated via complementation tests.
  • an integrase gene was synthesised with an increased GC content resulting in high level expression of HIV-1 IN in various mammalian cell lines.
  • the enzyme was shown to complement defective integrase carried by HIV-1 -derived vector particles and to act in trans on linear DNA substrates that are flanked by LTR fragments and encode a reporter gene.
  • Synthetic genes have been constructed in the past to optimize expression of eukaryotic genes in bacteria based on the knowledge that codon usage in prokaryotes is quite different from that in eukaryotes.
  • HIV (lentiviral) genes are not optimal for high level expression in eukaryotic cells. This is related to the mechanism HIV uses to circumvent the mRNA instability, namely Rev.
  • Rev mRNA instability
  • early mRNA transcripts will be spliced which results in expression of regulatory proteins such as Tat and Rev. Only late in the cycle, does Rev accumulation and Rev-RRE interaction block splicing and suppress AT-rich instability sequences resulting in unspliced transcripts encoding structural and enzymatic proteins.
  • the synthetic gene in accordance with the present invention clearly augments protein expression in mammalian cells, which is a prerequisite to detect the functionality of the enzyme in the cell
  • the presence of a gene with an increased GC content may well interfere with the mechanism of regulation of gene expression and be detrimental for viral replication.
  • a synthetic viral gene was designed for better optimized and more efficient expression in mammalian cells.
  • the HIV-1 integrase gene has a GC content of 40% whereas highly expressed human genes on average have a GC content of 55-61%.
  • the GC content is one aspect of the preferred nucleotide relationship or nucleotide pair frequency in accordance with the present invention.
  • the GC code content of a synthetic gene encoding HIV integrase would be increased up to 66% without altering the amino acid sequence.
  • this is not preferred in accordance with the present invention.
  • the choice among the alternative codons was biased in favour of preferred triplets (codons) found in a small group of genes of the total human genome which express well/strongly, e.g. human ⁇ -globin, -, ⁇ -actin and EF2 genes (method of determining the preferred codon usage).
  • the bias was such as to approximate the preferred nucleotide relationship or nucleotide pair frequency, i.e. within the range 53 to 63%o, more preferably 55-61% for the GC content. In fact a GC content of 59% rather than 66% was achieved.
  • the other rules for redesigning retroviral genes for eukaryotic expression are: (i) removal of potential splice sites, (ii) reduction of the number of CpG methylation sites, (iii) introduction of 5' and 3 '-untranslated regions (UTR) of a mammalian mRNA (in our case from human ⁇ -globin), (iv) addition of an extra N-terminal peptide (Met-Gly for the examples given below) for efficient initiation of translation.
  • UTR 5' and 3 '-untranslated regions
  • Method-Gly for the examples given below
  • a gene is provided to achieve high level expression of HIV-1 integrase in human cell lines by maintaining the amino acid sequence of IN from the pNL4-3 clone of HIV-1 while adapting the nucleotide codon usage to the codon usage of constitutively and highly expressed human genes ("preferred codon usage").
  • a first version of an artificial IN reading frame was based on random choice between alternative codons at each position using a random number generator, biasing in favour of preferred triplets as found in the human ⁇ -globin, ⁇ -, ⁇ -actin and EF2 genes.
  • the DNA sequence was substantially edited to remove potential splice sites and to reduce the number of CpG methylation sites, but keeping the overall GC content and codon usage close to optimal ("preferred nucleotide relationship" or "nucleotide pair frequency”).
  • the final version of the synthetic gene (Fig. 2 or SEQ ID NO: 1) contains fragments of the 5'- and 3 '-untranslated regions from the ⁇ -globin mRNA. This gene encodes for wild type HIV-1 integrase with addition of the N-terminal Met-Gly dipeptide. The extra glycine codon completes the Kozak's consensus sequence (ANNATGG) required for efficient initiation of translation.
  • the overall GC content is 59% compared to 40% in the wild type.
  • the gene was constructed from six synthetic DNA fragments, each approximately 150 bp long, by stepwise cloning. It should be understood that various homologs of the gene shown in Fig. 2 or SEQ ID NO:l are included within the scope of the present invention. Reapplication of the random number biasing procedure in accordance with the present invention would generate alternative sequences all of them coding for the same protein and all having a similar preferred nucleotide relationship or nucleotide pair frequency. All such synthetic gene homologs are included within the scope of the present invention.
  • the synthetic gene includes modification to those described above, the following modifications and improvements of the synthetic gene are included within the scope of the present invention.
  • the leader peptide can be replaced affecting the efficiency of translation and potential myristoylation (e.g. for example, a Met- Ala variant has been constructed).
  • the 5' and 3'-UTRs may be replaced by UTRs from other mammalian mRNAs to optimize the stability of the transcript.
  • Mutations in the open reading frame are also included within the scope of the present invention whereby the canonical integrase sequences (e.g. HHCC and DD35E) are preferably left unchanged.
  • a more soluble version can be made by introducing for example the F185K/ F185H mutations.
  • the present invention includes a variant synthetic gene with the D64V mutation, known to reduce drastically the enzymatic activity of integrase.
  • Synthetic genes of integrase are included within the scope of the present invention in which the genetic information of domains of other proteins are added. These domains preferably add additional properties to the enzyme such as sequence specificity in DNA binding. Examples of methods of providing specificity to a gene encoding integrase are described in WO 96/37626, US 5,811,270 without describing the specific innovative aspects of the present invention.
  • the synthetic gene for HIV-1 integrase was designed to circumvent inhibition of gene expression induced by instability sequences (INS) in the wild type integrase gene.
  • This approach can be applied to retroviral integrases in general.
  • the aforementioned design method may be used to redesign any retroviral viral gene encoding a protein with enzymatic activity for efficient expression in eukaryotes.
  • the design method of synthetic genes in accordance with the present invention will boost eukaryotic expression for retroviral genes encoding a protein with enzymatic activity, especially lentiviral integrases and pol proteins in general.
  • a low level transient expression from a wild type integrase gene with a downstream CTE of SRV-1 has been obtained by us using the methods of the present invention. Since the design of a synthetic gene in accordance with the present invention abolishes any need for co-expression of Rev and presence of RRE or CTE in the construct, this approach can improve expression of retroviral enzymes in general and integrases in particular.
  • various eukaryotic expression plasmids can be used. Expression can be under control of a constitutive promoter (for example hCMV and RSV) or an inducible promoter.
  • inducible expression systems examples include the ecdysone-inducible and the tetracyclin- inducible (Tet-Off and Tet-On) expression systems.
  • Tissue-specific promoters that limit expression in specific tissues may also be envisaged. Examples are the established neuron-specific promoters Thy-1 and enoiase.
  • Inducible promoters may limit cellular toxicity, although a cell line that stably expresses integrase was obtained.
  • expression may be induced at a desired moment using an inducible promoter or in desired tissues using a tissue-specific promoter.
  • the synthetic gene for integrase was cloned into the expression vectors pCEP4 and pBK-RSV under control of the human cytomegalovirus (hCMV) and Rous sarcoma virus (RSV) promoters, respectively.
  • hCMV human cytomegalovirus
  • RSV Rous sarcoma virus
  • Transient and stable expression of IN was obtained in both 293T and HeLa cell lines, as verified by immunoblotting (Fig. 1, 3) and indirect immunofluorescence (data not shown).
  • the expression levels from the hCMV promoter amounted to 10-20 ⁇ g of IN per 10 x 10 6 cells which is at least 25-fold higher than obtained with expression vectors that contain the unfused wild type HIV-1 integrase gene.
  • 293T-IN S Transfection of 293T cells with the episomal expression vector pCEP-IN followed by selection with hygromycinB, resulted in a stable cell line, referred to as 293T-IN S .
  • Indirect immunofluorescence staining revealed that 80-90% of selected cells produce integrase at detectable levels.
  • the expression level was about 0.5 ⁇ g of integrase per 10 x 10 6 cells.
  • the reduced cell growth kinetics of 293T-IN S (30-50%) as compared to the parental 293T cell line) is suggestive of cellular toxicity of integrase in mammalian cells. In HeLa cells integrase was found exclusively in the nuclei.
  • HIV-1 -derived lentiviral vectors have been developed by Naldini et al. and Zufferey et al. (Naldini et al. (1996), Science 272: 263-267; Zufferey et al. (1997), Nature Biotechnol. 15: 871-875).
  • Pseudotyped lentiviral vector particles are produced by transfecting 293T cells with a packaging plasmid encoding viral gag and pol proteins, a plasmid encoding the envelope of vesicular stomatitis virus and a plasmid encoding a reporter gene flanked by two long terminal repeats (LTRs).
  • the first generation packaging plasmid pCMV ⁇ R8.2, containing all HIV genes except for env and the transfer vector pHR'-CMVLacZ were used to produce wild type vector (WT vector).
  • Integrase-defective virus particles were produced using pCMV ⁇ R8.2IN(D64V) (Naldini et al.
  • C IN Complemented vectors
  • producer cells including pCEP-IN s , the expression vector containing the synthetic gene.
  • the transducing activity was restored up to 30% with C IN (Table 1). Complementation was due to stable integration, since an equal proportion of galactosidase-positive colonies was counted after multiple passages of the transduced cells.
  • the principle of trans-complementation of IN-defective virus was shown previously, using VPR-IN fusion expression constructs (Fletcher et al. (1997), EMBO J. 16: 5123-5138).
  • the transducing activity of catalytic domain mutants of EN was restored up to 20% by transcomplementation with VPR-IN.
  • a promoterless reporter substrate for measuring integrase activity in cell culture is based on this finding (Figs. 4A - C).
  • a method is proposed in which read-through transcription of the integrated promoterless reporter gene will occur when inserted within an actively transcribed region of the chromosome.
  • the construct designed is a linear DNA fragment, flanked by the 200 bp terminal fragments of the HIV LTRs that provide the integrase recognition sites.
  • the marker gene may encode luciferase, for instance.
  • IRES internal ribosome entry site
  • An embodiment of the present invention includes the construction of an efficient eukaryotic expression vector for a retroviral enzyme, e.g. HIV-1 integrase, based on the creation of a synthetic gene.
  • Expression from the eukaryotic expression vector can be transient after transfection of the plasmid in a eukaryotic cell by any of established transfection procedures. Expression may also be permanent in a cell line stably harbouring the expression vector.
  • An important aspect of the present invention and its applications is the functionality of an expressed retroviral enzymatically active protein, as opposed to mere the high level expression of an enzymatically inactive retroviral protein.
  • An embodiment of the present invention includes assays for evaluating integrase activity in cells transfected with a DNA substrate that is flanked by fragments of HIV LTR, a so-called mini-HIV. In both assays data point to enzymatic activity of IN.
  • DIAS detection of integrase activity through antibiotic selection
  • a resistance gene to a cytotoxic drug is present in the mini-HIV DNA.
  • the presence of IN in the transfected cell augments stable insertion of the resistance gene in the chromosome. Scoring is performed by comparing the residual number of colonies resistant to the cytotoxic agent in comparison with cells transfected with heterologous DNA.
  • a reporter gene without promoter is present downstream of an internal ribosome entry site (IRES) in mini-HIV (Fig. 4A).
  • IRS internal ribosome entry site
  • Fig. 4B The presence of IN in the transfected cell (Fig. 4B) augments stable insertion of the reporter construct in the host chromosome in close proximity to a cellular promoter (Fig. 4C). Scoring is performed by measuring enzyme activity expressed from the promoterless marker gene, e.g. luciferase.
  • the latter assay is highly amenable to evaluation of integrase inhibitors in cell culture in a microtiter plate format, adaptable for high throughput screening. Potential integrase inhibitors would result in the absence of or a significant decrease in the level of detectable signal from the promoterless marker gene.
  • Such an assay in accordance with the present invention involves screening test inhibitory compounds from large libraries of synthetic or natural compounds.
  • Synthetic compound libraries are commercially available from, for example, Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, NJ), Brandon Associates (Merrimack, NH) and Microsource (New Milford, CT).
  • a rare chemical library is available from Aldrich Chemical Company, Inc. (Milwaukee, WI).
  • libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from, for example, New Chemical Entities, Pan Laboratories, Bothell, WA or MycoSearch (NC), Chiron, or are readily producible. Plant extracts may also be obtained form the University of Ghent, Belgium. Additionally, natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means.
  • Cell lines that express integrase from a synthetic gene do have greater propensity to integrate foreign DNA, flanked by LTR fragments. These cell lines are thus more transducible.
  • An embodiment of this invention is the creation of eukaryotic cell lines (or cell culture systems) that are highly transducible (at least 200% compared to the parent cell).
  • Embodiments of the present invention also include applications in transgene technology to increase the efficiency of (non)homologous recombination in ES cells be it by transient expression from a plasmid or after induced expression of the retroviral integrase in ES cells transgenic for the synthetic gene.
  • the synthetic gene in accordance with the present invention may be brought into cells by any transfection agent or method (e.g. electroporation or lipofection) and may result in the stable integration of DNA in the chromosome.
  • Retroviral (lentiviral) vector packaging construct construct
  • integrase expressed from the synthetic gene in the producer cell can complement integrase-defective lentiviral virus particles encoded by a packaging plasmid and thus can substitute for the protein expressed by the packaging construct. It follows that in an expression vector based on one or more synthetic gene(s) for a lentiviral gag-pol gene, the synthetic gene(s) can substitute for the natural gene(s) in the packaging constructs resulting in Rev- independent high level protein expression.
  • the present invention includes a packaging construct based on non-lentiviral complex retroviruses in which protein expression is dependent on a Rev homologue such as Rex in the case of HTLV-I.
  • Lentiviral vectors per se capable of transducing a non-dividing cell, are known in the art (see Naldini et al. (1996), Science 272: 263-267, Zufferey et al. (1997), Nature Biotechnol. 15: 871- 875).
  • the vectors are plasmid-based or virus-based, and are configured to carry the essential sequences for incorporating nucleic acid, for selection and for transfer of the nucleic acid in the host cell.
  • Gag, pol and env genes of interest are known in the art.
  • a first vector can provide a nucleic acid encoding a viral gag and a viral pol
  • a second vector can provide a nucleic acid encoding a viral env gene product to produce a packaging cell.
  • Packaging cells or cell lines supply in trans the proteins necessary for producing infectious virions, themselves being incapable of packaging endogenous viral genomic nucleic acids (Watanabe & Temin (1983), Molec. Cell Biol. 3(12): 2241-2249; Mann et al. (1983), Cell 33:153-159; Embretson & Temin (1987), J. Virol. 61(9): 2675-2683).
  • the transfer vector into such packaging cells yields producer cells which release infectious particles carrying the foreign gene of interest.
  • Methods for transfection or infection are well known by those skilled in the art.
  • the recombinant vector is recovered from the culture media and titered by standard methods used by those of skill in the art.
  • the foreign or heterologous gene carried by the transfer vector can be any nucleic acid of interest which can be transcribed, but preferably is a nucleic acid encoding for a polypeptide of therapeutic benefit or of interest for gene therapy.
  • the env gene in the (second) packaging vector can be derived from any virus, including retroviruses, and is preferably amphotropic, allowing transduction of cells of human and other species, and is preferably under control of non-endogenous regulatory sequences.
  • Vectors can be made target-specific through linkage of the env protein with an antibody or a ligand for a particular receptor of a particular cell-type (cell-targeting).
  • gag-pol synthetic gene is based on a method to circumvent mRNA instability associated with these wild-type genes.
  • the method used by us to create an expression construct for high level and Rev independent eukaryotic expression of active HIV-1 integrase is employed.
  • Further construction of the vectors of the present invention, whereby natural gag-pol genes are replaced respectively by the synthetic genes of the present invention employ standard ligation and restriction techniques which are well understood in the art (see Maniatis et al, in Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory, N.Y., 1982).
  • Biosafety requires measurements to reduce the risk of generating recombinant replication competent retroviruses (RCR) as much as possible. Dividing the packaging functions into two genomes, one which express the gag and pol gene and another expressing the e «v gene product help to minimize the likelihood of generating RCR.
  • RCR replication competent retroviruses
  • US5,994,136 describes the production of lentiviral vectors with an even more remote possibility of generating replication competent lentiviruses by functionally deleting the tat gene, which is encoding for a regulating protein that promotes viral expression through a transcriptional mechanism.
  • the likelihood of recombination between the transfer vector, that still contains natural genetic information of the lentivirus like part of the gag gene, and synthetic packaging genes will be considerably reduced, further improving the biosafety of the lentiviral vectors.
  • DNA sequence mismatching, as induced by the replacement of nucleotides in the third position compared to the natural gene, seems to present a considerable barrier to homologous recombination in a wide variety of species. It is therefore also unlikely that contaminating or endogenous HIV virus particles would exchange the natural integrase gene for the synthetic one through recombination.
  • the open reading frame of IN from the HIV-1 clone HXB2 was PCR amplified using Pfu DNA polymerase (Stratagene, Cambridge, UK) with the primers
  • the absence of mutations was verified by DNA sequencing.
  • the RRE sequence of HIV-1, clone HXB2 was PCR amplified using the primers 5'-TTCCGCTCG C7TAGCACCCACCAAGGCAAAG C7 and 5'-TCGCGGATCCAAGGCACAGCAGTGGTGCAAATG.
  • the PCR fragment was subcloned in the sense orientation downstream of the integrase gene in pCEP-I to produce pCEP-IN-RRE.
  • the CTE sequence (obtained from plasmid pS12; Tabemo et al., 1996, J. Virol. 70: 5998-6011) was cloned in pCEP4 in the correct orientation, followed by the insertion of the integrase gene upstream of the CTE. This resulted in the plasmid pCEP-IN-CTE.
  • the construction of pGFP-IN is explained in Pluymers et al. (1999), (Virology 258: 327-332).
  • the Rev expression plasmid, pEF321-cREV was provided by Sandozmaschines Institut, Vienna, Austria.
  • PCR amplification and plasmid construction employed standard techniques like standard ligation and restriction techniques and conditions which are well understood in the art (see Maniatis et al, in Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratroy, N.Y., 1982).
  • the restriction sites Nhel, Pstl, BamHI, Nael, Narl (indicated in Fig.2 ) divide the sequence of the synthetic gene into 6 fragments each approximately 150 bp long that correspond to the sequences 1-149, 144-306, 301-456, 451-623, 618-776, 771-930 (Fig. 2).
  • Each of the fragments was constructed separately by annealing and extending two partially complementary oligonucleotides (85-95 nt long, PAGE-purified and 5'- phosphorylated, synthesized by Gibco BRL Life Technologies, Merelbeke, Belgium) using Sequenase (Amersham-Pharmacia, Buckinghamshire, UK) .
  • pCEP4 is an episomal mammalian expression vector containing the human cytomegalovirus (hCMV) immediate early enhancer/promoter.
  • Epstein Barr virus replication origin (oriP) and nuclear antigen (encoded by the EBNA-1 gene) permit extrachromosomal replication in human, primate and canine cells.
  • a hygromycin resistance gene is present, permitting selection of stably transduced clones by hygromycinB (GIBCO BRL).
  • the same 1 kb fragment was also cloned between Nhel and Xhol sites of the pBK-RSV expression vector (Stratagene) (the Nhel cohesive end of the vector DNA was filled in using T4 DNA polymerase) resulting in pRSV-IN s .
  • IN S gene is driven by the promoter of Rous sarcoma virus (RSV).
  • RSV Rous sarcoma virus
  • the presence of the neomycin resistance gene allows selection of stably transduced clones by geneticin (G418) (GIBCO BRL).
  • the DNA substrate for the DIPR assay was obtained by linearization of pLTR- IRES-Luc with Seal.
  • This plasmid was constructed in the following way. First, the 350 bp Kpnl EcoRI fragment of pU3U5 (Cherepanov et al.(1999), Nucleic Acids Research 27: 2202-2210) containing the terminal U3 and U5 regions of the HXB2 HIV-1 LTRs was cloned between the Kpnl and EcoRI sites of pUC19 resulting in pUC-LTR.
  • HeLa and 293 cells were obtained from American Type Culture Collection. HeLa and 293 cells were grown in Dulbecco's modified Eagle's medium (DMEM) (GibcoBRL) supplemented with 10 % FCS, 0.12 % (v/w) sodium bicarbonate (GibcoBRL), 2 mM glutamine (GibcoBRL) and 20 ⁇ g/ml gentamycin (GibcoBRL) at DMEM (DMEM) (GibcoBRL) supplemented with 10 % FCS, 0.12 % (v/w) sodium bicarbonate (GibcoBRL), 2 mM glutamine (GibcoBRL) and 20 ⁇ g/ml gentamycin (GibcoBRL) at DMEM (GibcoBRL) at DMEM) (GibcoBRL) supplemented with 10 % FCS, 0.12 % (v/w) sodium bicarbonate (GibcoBRL), 2 mM glut
  • SV40 large T antigen express SV40 large T antigen and were grown in DMEM (GibcoBRL) with glutamax supplemented with 10% fetal calf serum, 45 U/ml penicillin G (Serva, Heidelberg, Germany) and 45 ⁇ g/ml streptomycin sulphate (Sigma-Aldrich, Bornem, Belgium).
  • 293 and 293T cells were transfected using polyethylenimine (PEI) (Abdallah et al. (1997), Hum. Gene Therapy 7:1947-1954). Polyethylenimine Mw ⁇ 25.000 was from Sigma-Aldrich (Bornem, Belgium). Cells were grown to 50-70 %> confluency in DMEM with glucose, glutamax and 10 % fetal calf serum (FCS) (Gibco BRL). Medium was replaced by medium containing 1 % FCS 3 hours before transfection. Mixture of DNA and PEI was added to cells in a minimal volume of medium. Next day the medium was changed to DMEM containing 25 mM HEPES. Transformation efficiency obtained in this way was 50-80 %.
  • PEI polyethylenimine
  • FCS fetal calf serum
  • HeLa cells were routinely transfected by electroporation. The cells were first trypsinized at 80 % confluency and pelleted by low speed centrifugation. The cells were then resuspended at a density of 2 x 10 6 cells/ml in growth medium; 0.5 ml of this solution was aliquoted into 4 mm cuvettes (Eurogentec, Seraing, Belgium) and 20 ⁇ g DNA was added to the cell suspension. After the electric pulse (10 ⁇ F, 250 V), cells were allowed to rest for 10 min at room temperature before dilution into growth medium..
  • Detection limit was 0.1-0.5 ng of recombinant integrase.
  • Total protein concentration was determined on cells lysed with 1%SDS/1 mM PMSF (Sigma), using the BCA protein assay (Pierce, Illinois USA). For western blot analysis 10 ⁇ g of total protein was evaluated.
  • DIPR promoterless reporter gene
  • 293T and 293T-IN S cells were seeded in six- well plates at a density of 10 6 cells/well 24 hr before transfection. Five ⁇ g of DNA was transfected per well using PEI. 48 hr post-transfection, 5 x 10 5 cells were lysed to determine the luciferase activity using the Luciferase Assay SystemTM (Promega Benelux, Leiden, The Netherlands) and the LumicountTM (Packard, Meriden, CT). The protein concentration of the lysate was determined using the Bradford method (Bio-Rad protein assay, Bio-Rad, Hercules, CA). The relative luciferase activity was calculated by dividing the luminescence values by the protein concentration.
  • Lentiviral vectors Lentiviral vector production HIV-1 -derived vector particles, pseudotyped with the envelope of vesicular stomatitis virus (VSV), were produced by transfecting 293T cells with a packaging plasmid encoding viral gag and pol proteins (pCMV ⁇ R8.2), a plasmid encoding the envelope of vesicular stomatitis virus (pMDG) and a plasmid encoding a reporter gene flanked by two long terminal repeats (LTRs) (pHR'-CMVLacZ).
  • the first generation packaging plasmid, containing all HIV genes except for env, and the transfer vector were a kind gift from Dr. O.
  • the vector particles were sedimented by ultracentrifugation in a swinging-bucket rotor (SW27 Beckman, Palo alto, CA) at 25, 000 rpm for 2 hr at 4°C. Pellets were redissolved in PBS resulting in a 100-fold concentration. Different viral stocks were normalized based on p24 antigen content (HIV-1 p24 Core Profile ELISA, DuPont, Dreieich, Germany) for use in complementation assays.
  • Integrase-defective virus particles were produced using pCMV ⁇ R8.2IN(D64V), obtained from Dr. D. Trono, (Geneva, Switzerland) as packaging plasmid (Naldini et al. (1996), Science 272: 263-267).
  • Complemented vectors were produced by expressing integrase from pCEP-IN s in 293T cells after quadruple transient transfection. Vector preparations were normalized for p24 antigen count Vector was added to target cells in the presence of 2 ⁇ g/ml polybrene and left overnight. After removal of vector, cells were incubated for an additional 36 hrs.
  • Transduction efficiency was determined by coimting the number of blue cells 48 hrs after infection in one of the wells, whereas the cells in the duplicate well were splitted 1:2. Half of the sample remained in the well and was stained at confluency (passage 1) whereas the other half was cultured in a 48-well plate. At confluency, these cells were again splitted 1:2. Finally, cells were brought in a 24-well plate and grown to confluency (passage 3, dilution 1:8). After staining, the efficiency of stable transduction was measured by counting blue colonies.
  • C 293T and 293T-IN S were transfected with linearized pLTR-IRES-Luc and 2 ⁇ g of pCMV-IN s .

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Abstract

La présente invention concerne un gène synthétique ou une région de gène ayant une utilisation améliorée de codon par rapport au gène de type de sauvage et servant à l'expression de niveau élevé d'une protéine rétrovirale dans des cellules eucariotes, la protéine rétrovirale exprimée possédant une activité enzymatique dans la cellule eucariote. L'invention concerne également un gène synthétique ou une région de gène codant une enzyme rétrovirale ou une partie d'enzyme exprimée normalement dans une cellule mammalienne ou dans une autre cellule eucariote, au moins un codon non préféré dans le gène de type sauvage codant l'enzyme ayant été remplacé par un codon préféré codant le même acide aminé. La protéine rétrovirale peut être une protéase, une transcriptase inverse, une protéine intégrase ou son précurseur gag-pol de polyprotéine. Dans un mode de réalisation la protéine rétrovirale à activité enzymatique est une protéine lentivirale. Dans d'autres modes de réalisation, la protéine à activité enzymatique est une enzyme pol. Dans des modes de réalisation préférés, la protéine à activité enzymatique est une intégrase lentivirale, ou, mieux, une enzyme de VIH; dans d'autres modes de réalisation, la protéine à activité enzymatique est idéalement une intégrase de VIH. L'invention concerne également un procédé de détection de l'intégrase intracellulaire au moyen d'un gène reporter démuni de promoteur.
PCT/EP2000/003765 1999-04-26 2000-04-26 Gene synthetique destine a l'expression d'une proteine retrovirale active dans les eucariotes WO2000065076A2 (fr)

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IL14609000A IL146090A0 (en) 1999-04-26 2000-04-26 A synthetic gene for expression of a retroviral protein with wild type activity in eukaryotic cells
CA002369058A CA2369058A1 (fr) 1999-04-26 2000-04-26 Gene synthetique destine a l'expression d'une proteine retrovirale active dans les eucariotes
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AU45579/00A AU778106B2 (en) 1999-04-26 2000-04-26 Synthetic gene for expressing active retroviral protein in eukaryotes
JP2000614410A JP2002542791A (ja) 1999-04-26 2000-04-26 真核生物中で活性を有するレトロウイルスタンパク質を発現させるための合成遺伝子
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WO2001046408A3 (fr) * 1999-12-23 2001-12-13 Us Gov Health & Human Serv Clones moleculaires avec genes mutes vih gag/pol, vis gag et vis env
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US5468629A (en) * 1993-04-13 1995-11-21 Calhoun; Cornelia Method of promoting in vitro homologous recombination transfection in mammalian cells using the RecA protein
US5434065A (en) * 1993-05-06 1995-07-18 President And Fellows Of Harvard College In vivo selection of microbial virulence genes
US5811270A (en) * 1994-05-20 1998-09-22 Grandgenett; Duane P. In vitro method for concerted integration of donor DNA molecules using retroviral integrase proteins
US6200811B1 (en) * 1996-04-02 2001-03-13 The Regents Of The University Of California Cell transformation vector comprising an HIV-2 packaging site nucleic acid and an HIV-1 GAG protein
US6114148C1 (en) * 1996-09-20 2012-05-01 Gen Hospital Corp High level expression of proteins
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US6656706B2 (en) 1999-12-23 2003-12-02 The United States Of America As Represented By The Department Of Health And Human Services Molecular clones with mutated HIV gag/pol, SIV gag and SIV env genes
US8076100B2 (en) 1999-12-23 2011-12-13 The United States Of America As Represented By The Department Of Health And Human Services Molecular clones with mutated HIV gag/pol, SIV gag and SIV env genes
EP2128256A3 (fr) * 1999-12-23 2009-12-23 The Government Of The USA, As Represented By The Secretary, Dept. Of Health & Human Services Clones moléculaires avec gènes mutés VIH GAG/POL, VIS GAG et VIS ENV
WO2001062958A1 (fr) * 2000-02-23 2001-08-30 National Cancer Centre Of Singapore Pte Ltd. Evaluation de la signification fonctionnelle d'une sequence de nucleotides consistant a soumettre les donnees du cadre de lecture a un traitement de donnees
WO2001079518A3 (fr) * 2000-04-19 2002-05-16 Oxford Biomedica Ltd Procede
EP2194137A3 (fr) * 2000-04-19 2010-07-28 Oxford BioMedica (UK) Limited Cellules comprenant des particules rétrovirales avec des codons optimisés
EP1402019A4 (fr) * 2001-06-01 2006-04-19 Us Gov Health & Human Serv Clones moleculaires contenant des genes mutes hiv gag/pol, siv gag et siv env
US8133494B2 (en) 2001-07-05 2012-03-13 Novartis Vaccine & Diagnostics Inc Expression cassettes endcoding HIV-1 south african subtype C modified ENV proteins with deletions in V1 and V2
US6835568B2 (en) 2001-10-30 2004-12-28 Virxsys Corporation Regulated nucleic acid expression system
US7572629B2 (en) 2003-05-06 2009-08-11 National Institute Of Advanced Industrial Science And Technology Multiple gene transcription activity assay system
US8492522B2 (en) 2005-10-07 2013-07-23 Istituto Di Ricerche Di Biologia Molecolare P. Angeletti Spa Matrix metalloproteinase 11 vaccine
GB2535563A (en) * 2014-09-11 2016-08-24 Jnc Corp Design method for synthetic genes
US9938539B2 (en) 2014-09-11 2018-04-10 Jnc Corporation Method for synthetic genes
US10196650B2 (en) 2014-09-11 2019-02-05 Jnc Corporation Method for synthetic genes
GB2535563B (en) * 2014-09-11 2020-10-28 Jnc Corp Design method for synthetic genes
US11268106B2 (en) 2014-09-11 2022-03-08 Jnc Corporation Method for synthetic genes

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BR0010077A (pt) 2002-01-15
JP2002542791A (ja) 2002-12-17
NO20015168L (no) 2001-12-14
RU2001131728A (ru) 2004-02-20
AU778106B2 (en) 2004-11-18
NO20015168D0 (no) 2001-10-23
IL146090A0 (en) 2002-07-25
AU4557900A (en) 2000-11-10
HUP0201019A2 (en) 2002-08-28
PL351988A1 (en) 2003-07-14
CA2369058A1 (fr) 2000-11-02
WO2000065076A3 (fr) 2001-12-13

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