WO2012152632A1 - Système de vecteur rétroviral minimal - Google Patents

Système de vecteur rétroviral minimal Download PDF

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Publication number
WO2012152632A1
WO2012152632A1 PCT/EP2012/058057 EP2012058057W WO2012152632A1 WO 2012152632 A1 WO2012152632 A1 WO 2012152632A1 EP 2012058057 W EP2012058057 W EP 2012058057W WO 2012152632 A1 WO2012152632 A1 WO 2012152632A1
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vector
expression
vector system
retrovirus
transfer
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PCT/EP2012/058057
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German (de)
English (en)
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Dirk Lindemann
Erik MÜLLERS
Sylvia KAULFUß
Kristin Stirnnagel
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Technische Universität Dresden
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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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/17011—Spumavirus, e.g. chimpanzee foamy virus
    • C12N2740/17041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/17043—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
    • 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
    • C12N2740/17011—Spumavirus, e.g. chimpanzee foamy virus
    • C12N2740/17051—Methods of production or purification of viral material
    • C12N2740/17052—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles

Definitions

  • the present invention relates to a retrovirus vector system having a
  • Transfer vector containing at least one expression cassette for receiving a nucleotide sequence coding for a heterologous, in particular non-retroviral, gene product with respect to the relevant retrovirus, which is characterized in that it contains no sequences of the relevant retrovirus.
  • Objects of the present invention relate to host cells associated with the
  • Methods for producing the host cells a kit for expression of a relative to the relevant retrovirus heterologous, in particular non-retroviral gene product containing the vector system, and a method for the expression of a relative to the respective retrovirus heterologous, in particular non-retroviral gene product in a cell using the vector system.
  • Retroviruses pack two copies of their RNA genomes (+ stranded orientation, single-stranded, therefore the same structure as cellular mRNA) during the
  • RNA generation in the viral capsid During their replication cycle, these packaged RNA genomes are converted into a single copy of double-stranded (ds) DNA by retroviral reverse transcriptase (RT) and subsequently integrated into the host cell genome through a process that is dependent on the viral integrase (IN) protein.
  • RT retroviral reverse transcriptase
  • I viral integrase
  • Orthotrophic viruses e.g., murine leukemia virus (MLV), human immunodeficiency virus (HIV)
  • MMV murine leukemia virus
  • HSV human immunodeficiency virus
  • FV Foamy viruses
  • FV particles containing full-length viral dsDNA genomes are responsible for the majority of infection events.
  • the integration of retroviral genomes into the host cell genome leads to long-term expression in the infected cells. If no integration occurs, two different types of transient retroviral protein expression are known ( Figure 2). First, if reverse transcription continues to take place, but the retroviral integrase-mediated integration is prevented, substantially transient transcription from unintegrated, episomal viral DNA genomes is observed. These episomal viral DNA genomes can enter the host cell genome through nonhomologous cells at very low frequency
  • Integrate recombination events (similar to a stable plasmid transfection), and lead to a long-term expression in these cells. Second, if reverse transcription does not take place, the two copies of the packaged viral RNA genomes can be translated as they are released in the cytoplasm. In general, the first mechanism leads to a longer and stronger protein translation than the second mechanism.
  • retroviral gene delivery systems for transient transgene expression ( Figure 2).
  • RV integration-deficient retroviral vectors
  • ⁇ systems which contain an enzymatically inactivated retroviral integrase
  • the transgene expression caused by such an RV decreases steadily over a period of 10-14 days in dividing cells, but may also be stable over weeks in quiescent cells.
  • low long-term transgene expression due to non-virally mediated non-homologous recombination of viral and cellular DNA genomes can be detected, resulting in stable integration of the viral genome into the host cell genome.
  • PMT particle-mediating mRNA transfer
  • PMT-mediated transgene expression is completely transient and no non-homologous recombination events of viral and cellular nucleic acids have been observed, which could result in long-term genetic modification.
  • the level and duration of PMT-mediated transgene expression is significantly lower and shorter than that of ⁇ -RV systems.
  • the reason for this is that PMT-mediated transgene expression originates from the two copies of the viral RNA genome per virus particle delivered into the cytoplasm of the target cell, while episomal delivery of one copy of the reverse transcribed viral DNA genome per virus particle ⁇ -RV systems for generating multiple mRNA copies due to de novo transgene transcription by the cellular transcriptional machinery and therefore to the amplification of the
  • RNA stem-loop structures RNA stem-loop structures
  • WO-A-2010/1 1 1608 describes a foamy virus vector system in which at least one recombinant vector comprises an expression sequence encoding at least one component of a foamy virus particle, wherein at least one codon of the expression sequence is optimized for expression in Homo sapiens is.
  • a retroviral vector system is provided according to the invention, which (a) one or more recombinant vector (s), the /
  • a transfer vector comprising at least one expression cassette for receiving a gene product heterologous to the retrovirus, preferably a non-retroviral gene product containing nucleotide sequence encoding, wherein the transfer vector has no sequences of the respective retrovirus.
  • Packaging cell line provided via a suitable transfer vector
  • the present invention also generally provides a minimal retroviral vector system in that (1) one or more
  • Recombinant vector containing only those expression sequences coding for the Env and Gag proteins of the retrovirus with respect to the virus in question, and (2) comprising a (random) transfer vector comprising at least one expression cassette for receiving a contains nucleotide sequence coding for a gene product.
  • the transfer vector may also contain retroviral sequences, such as c / s-active packaging sequences.
  • Suitable retroviral vector systems according to the present invention are, for example, HIV-1, MLV and foamy virus vector systems.
  • the vectors or the vector according to component (a) of the vector system according to the invention represents or provide expression in a suitable
  • the components (a) also Pol and / or other retroviral proteins, such as in HIV-1, for example, one or more of the proteins Tat, Rev, Vpr and Vpu be provided.
  • a particularly preferred embodiment of the present invention is a foamy virus vector system comprising (a1) a recombinant vector containing a
  • the present invention is based on the surprising discovery that, contrary to the teachings of the prior art, in a retroviral vector system such as HIV-1, MLV or foamy virus vector systems
  • Transfer vectors can be used which do not contain any of the relevant retrovirus such as a foamy virus derived nucleotide sequences, in particular no c / s -effective sequences, such as, for example, in the case of the foamy CAS I / CAS II, and to an exclusively transient expression of a Transgene can be used in corresponding target cells.
  • a foamy virus derived nucleotide sequences in particular no c / s -effective sequences, such as, for example, in the case of the foamy CAS I / CAS II, and to an exclusively transient expression of a Transgene can be used in corresponding target cells.
  • a transfer vector which does not contain sequences of the relevant retrovirus, in particular a transfer vector whose
  • Nucleotide sequence is designed such that it has a sequence window size of about 100 or more nucleotides homology to a nucleotide sequence of the relevant retrovirus, preferably any retrovirus, of at most 50%, preferably at most 40%, more preferably at most 10% or less ,
  • PV prototypical FV
  • HBV human FV
  • Foamy virus vector system according to the invention or a recombinant vector (component (a2) of the foamy virus vector system of the present invention) containing / containing corresponding expression sequences must be provided to detect infectious particles which are above the inventive Transfer RNA provided RNA to produce.
  • component (a2) of the foamy virus vector system of the present invention containing / containing corresponding expression sequences must be provided to detect infectious particles which are above the inventive Transfer RNA provided RNA to produce.
  • embodiments of the present invention may be advantageous in providing an expression sequence for a polymorphic protein of a foamy virus.
  • a pol expression sequence can be provided on one or both of the vectors according to component (a1) and / or on the vector according to component (a2).
  • a further recombinant vector (c) may be provided which comprises a
  • Pol protein of foamy virus Contains expression sequence coding for a Pol protein of foamy virus.
  • the respective expression sequence for the pol protein is genetically biosynthetically inactivated.
  • the invention envisages one or both of the
  • the foamy virus vector system of the present invention is preferably derived from a human foamy virus (HFV or also PFV, see above), but can also be derived from monkey isolated foamy viruses (SFV) or other mammals isolated foamy viruses or hybrids or based on such.
  • HBV human foamy virus
  • SFV monkey isolated foamy viruses
  • Particularly preferred transfer vectors of the present invention have a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2.
  • the vector according to SEQ ID NO: 1 is also referred to as vector pczi and was determined on the basis of the pcDNA3.1 + zeo vector (Invitrogen) and the CMV intron A of the pHIT vectors (Soneoka et al. (1995) Nucleic Acids Res. 4), 628-633) (see Heinkelein et al., (2002) J. Viral., 76 (9), 3774-3783).
  • the vector according to SEQ ID NO: 2 is also referred to as vector pSFFVU3 and is based on the pEGFP vector (Clontech) in which the CMV promoter has been replaced by an SFFV U3 promoter variant in which a 42 bp duplication
  • a "gene product” according to the present invention may be any product which is expressible via an expression cassette in a transfer vector defined in the present disclosure in suitable target cells Transcription product derived expression products such as in particular translation products or products resulting from a processing of the immediate gene product.
  • a nucleotide sequence inserted into an expression cassette of a transfer vector of the present invention may comprise peptides or proteins, in particular of non-retroviral origin, in particular heterologous peptides and / or proteins (eg a non-foamy virus protein) with respect to the respective retrovirus. encode.
  • peptides or proteins in particular of non-retroviral origin, in particular heterologous peptides and / or proteins (eg a non-foamy virus protein) with respect to the respective retrovirus. encode.
  • RNAs Transcription products that are not translated into peptides / proteins in cells. Examples are tRNAs. Other non-peptidic and non-proteinaceous gene products are antisense RNAs, siRNAs, shRNAs, miRNAs (also known as microRNAs), pre-miRNAs and pri-miRNAs.
  • a nucleotide sequence which codes for at least one heterologous, in particular non-retroviral protein is preferably inserted into the expression cassette.
  • Nucleotide sequence may also at least partially encode a reporter protein or peptide. Suitable reporter proteins or peptides are in the state of
  • the reporter protein may be a GFP protein or a derivative thereof such as EGFP and similar fluorescence-generating proteins.
  • expression sequence means according to the invention a
  • a foamy virus protein such as Gag, Env or Pol
  • suitable promoter and terminator sequences such that the protein is expressed in a suitable host cell.
  • Other functional sequences that may be associated with the expression sequence are enhancer sequences, IRES sequences, and other functional elements known to those skilled in the art that are useful for directing the expression of a protein in a host cell.
  • a transfer vector according to the invention in which one coding for at least one gene product
  • the above embodiments also apply to the transfer vector with respect to the expression cassette (ie without inserted nucleotide sequence coding for at least one gene product encoded) or the expression sequence (ie with inserted
  • Nucleotide sequence encoding at least one gene product of operably linked functional sequence elements, respectively.
  • Expression cassette is thus a nucleotide sequence containing expression-controlling elements as set forth above and is preferably equipped with a multiple cloning site (MCS) and / or homologous sequences
  • a further subject of the present application is a host cell transfected with a vector system of the present invention.
  • Suitable host cells are, for example, bacterial cells such as E. coli in order to propagate the vector system according to the invention and / or its components.
  • Other preferred host cells are those designed to produce and deliver retroviral particles by expressing at least the essential viral proteins via the vector (s) of component (a) or component (1) of the vector systems of the present invention.
  • the preferred host cells are, for example, those for the production and discharge of foamy virus particles by expression of at least the gag and Env proteins of a foamy virus via the vectors according to (a1) or the vector according to (a2) are designed.
  • Examples of such cells are in particular cell lines such as human 293 or 293T cells and primate cell lines such as Cos1 or Cos7 cells.
  • Such host cells serve the production of infectious virus particles or virus-like particles (English, virus-like particles, VLPs), which contain the genetic information for the expression of the desired gene product and when introduced into with retrovirus particles ( Virionen) transduzierbare cells for the expression of the transfer vector coded
  • mammalian, avian, reptile and amphibian cells For example, mammalian, avian, reptile and amphibian cells.
  • Preferred cells for infection with retroviral particles such as foamy virus particles are human cells such as the cell line HT1080 used in the examples below.
  • a kit comprises for the expression of a gene product according to the invention, such as, for example, a heterologous, preferably non-retroviral, protein, such as a non-foamy virus protein, with respect to the respective retrovirus
  • the kit according to the invention also comprises (iii) cells which are suitable for infection with retrovirus particles, e.g. Foamy virus particles are suitable.
  • retrovirus particles e.g. Foamy virus particles are suitable.
  • the kit according to the invention can thus also contain host cells (packaging cells) as defined above together with the cells suitable for infection with retrovirus particles.
  • a further subject of the present invention is also a method for expressing the respectively desired gene product, such as e.g. a heterologous, especially non-retroviral, such as non-foamy virus, protein in a cell containing the steps
  • the vector (s) due to expression of at least the essential viral proteins via the vector (s) according to component (a) and (1) of the vector systems of the invention (e.g., Gag and Pol
  • Suitable conditions and culture media according to step (B) are known to a person skilled in the art and can be described, for example, in the latest issue of Ausubel et al. (Ed.) "Current Protocols in Molecular Biology", Wiley, New York. In this regard, for foamy virus systems of the present invention
  • a further subject matter of the present invention relates to a method for expressing a gene product as defined herein, preferably a non-retroviral gene product such as a non-retroviral, in particular non-foamy virus protein in a host organism, comprising introducing a host cell as defined above (US Pat. Packaging cell), which is used to form and
  • retrovirus particles eg foamy virus particles
  • at least the essential virus proteins via the vector (s) according to component (a) or (1) of the vector systems according to the invention eg gag and pol Proteins of a foamy virus via the vectors according to (a1)
  • retrovirus particles such as e.g. Foamy virus particles is / are transduable.
  • the host organism may be a human or non-human, in particular
  • the gene product such as a heterologous gene product, especially a non-retroviral, preferably non-retroviral Foamy virus protein in the host organism can be advantageously used to treat a genetic defect of the host organism.
  • a protein not or insufficiently expressed in the host organism due to a genetic defect to be sufficiently rich in the host organism by expression via the vector system of the present invention
  • Embodiments of the invention may be performed by expression of RNAi-triggering gene products (e.g., siRNA, shRNA, miRNA, pre-miRNA, pri-miRNA) in the RNAi-triggering gene products (e.g., siRNA, shRNA, miRNA, pre-miRNA, pri-miRNA) in the RNAi-triggering gene products (e.g., siRNA, shRNA, miRNA, pre-miRNA, pri-miRNA) in the
  • a gene regulation can be carried out in order to downregulate, for example, a gene associated with a disease.
  • Fig. 1 shows a schematic representation of various retroviral
  • Fig. 2 shows a schematic representation of different retroviral
  • PFV vectors were prepared using an expression-optimized 4-component PFV vector system.
  • This vector system consists of the transfer vector puc2MD9, which contains an internal SFFV U3 driven EGFP expression cassette, and the packaging constructs pcoPE (Env), pcoPG4 (Gag) and pcoPP (Pol).
  • the vector particles differ in the type of cotransfected Pol packaging construct.
  • Wt pcoPP (wt, all enzymatic activities intact); pcoPPI (iPR, enzymatically inactivated protease); pcoPP2 (iRT, enzymatically inactivated reverse transcriptase); pcoPP3 (ilN, enzymatically inactivated integrase).
  • HT1080 target cells were transduced with undiluted viral vector supernatant and EGFP expression in the cell populations by means of
  • Viral particles with enzymatically inactivated protease (iPR) or reverse transcriptase (iRT) enable weak and short lasting transient EGFP expression.
  • Particles with an enzymatically inactivated integrase (ilN) lead to a stronger but largely transient EGFP expression whereas wild-type particles lead to a strong constitutive transgene expression.
  • Figure 12 shows the characteristics of various embodiments of PFV transfer vector constructs of the present invention.
  • FIG. 1 Schematic representation of the transfer vectors pMD9, SFFV U3 EGFP PFV3 'and the vector SFFV U3 EGFP according to the invention.
  • pMD9 standard PFV transfer vector with an internal SFFV-U3 driven EGFP transgene cassette and essential PFV cis-containing packaging sequences (CAS I, CAS II);
  • SFFV U3 EGFP PFV 3 ' contains SFFV U3-driven EGFP expression vectors of the 3' parts of the PFV vector genome but has no c / s-active packaging sequences;
  • SFFV U3 EGFP SFFV U3-driven EGFP expression vector containing no PFV sequences.
  • Particulate release and pol packaging was checked by Western blotting of particle and cell lysates.
  • Nucleic acid composition was determined by qPCR. Shown are the respective values, starting from the respective values
  • Transfer vectors shows the influence of transgenic transcriptional control elements on RNA transfer efficiency.
  • PFV vectors were prepared by transient transfection of 293T cells using a codon-optimized 4-component PFV vector system according to WO-A-2010/11608 using the transfer vectors of (A). Subsequently, HT1080 target cells with undiluted viral vector supernatant or a
  • Transduced 1 10 dilution thereof and measured EGFP transgene expression by time-dependent flow cytometry.
  • PFV vectors were prepared by transiently transfecting 293T cells using an expression-optimized 4-component PFV vector system according to WO-A-2010/11608 using the pcziEGgP transfer vector of the present invention as described in FIG. Subsequently, HT1080 target cells were transduced with undiluted virus vector supernatant and the EGFP transgene expression was measured time-dependent by flow cytometry.
  • Retroviral vector systems derived from various retrovirus species. Retroviral vectors were generated by transient transfection of 293T cells using a codon-optimized 4-component PFV vector system (PFV) or conventional mouse leukemia virus (MLV) or human
  • HIV Immunodeficiency virus
  • transfer vectors of the present invention for transient (A) or stable (B) transgene expression generated.
  • HT1080 target cells with undiluted viral vector supernatant in the case of the transient transfer vectors (A) or a series of 10-fold dilutions for the stable transfer vectors (B) were transduced and the EGFP transgene expression was measured by flow cytometry.
  • Transfer vector supernatants were calculated from day 3 post infection d3 flow cytometry data.
  • Retroviral vectors were prepared by transient transfection of 293T cells using various combinations of packaging plasmids and transfer vectors for stable (MD9 qP) or transient marker gene expression (EGqP). Packing constructs for wild-type gag (Gag wt), pol (Pol wt) and Env (Env wt) as well as pol variants with enzymatically inactivated RT domain (Pol iRT) or integrase domain (ILN) and Env variants for fusion-inactive envelope protein ( Env iSU / TM) in single combinations.
  • HT1080 target cells were serially transduced with serially diluted vector supernatant and the EGFP transgene expression was measured time-dependently by flow cytometry.
  • Packaging plasmid was replaced with pUC19 DNA.
  • RNA transfer-mediated Cre recombinase expression in R26R-YFP mouse embryo fibroblasts PFV vector supernatants were carried through Transient transfection of 293T cells using an expression-optimized 4-component PFV vector system according to WO-A-2010/11608 using various retroviral (MD9 qP, MD9 Cre) or RNA (EGqP, Cre) transfer vectors of the present invention Invention as described in FIG. 3 described.
  • FIG. 10 Course of the transient EGFP expression in the period from 1 to 72.
  • PFV vectors were generated by transient transfection of 293T cells using a
  • the vector particles also differ in the nature of the cotransfected Pol packaging construct.
  • Wt pcoPP (wt, all enzymatic activities intact); pcoPP2 (iRT, enzymatically inactivated reverse transcriptase); pcoPP3 (ILN, enzymatically inactivated integrase).
  • HT1080 target cells with undiluted virus vector supernatant were transduced by spinoculation (30 min, 10 ° C, 1200g) at various time points after seeding and EGFP transgene expression of the various samples harvested at the same time was measured by flow cytometry.
  • A Schematic overview of the time course of the experiment.
  • B Mean EGFP fluorescence intensity (EGFP MFI) of the various target cell samples after transduction with undiluted cell-free 293T supernatant as a function of time point
  • PFV vectors were generated by transient transfection of 293T cells using a
  • pcoPP wt, all enzymatic activities intact
  • pcoPP2 iRT, enzymatically inactivated reverse transcriptase
  • pcoPP3 ILN, enzymatically inactivated integrase
  • HT1080 target cells with undiluted viral vector supernatant were transduced by spinoculation (30 min, 10 ° C, 1200g) and EGFP transgene expression was measured time-dependently by flow cytometry.
  • A Schematic overview of the time course of the experiment.
  • B Mean EGFP fluorescence intensity (EGFP MFI) of the various target cell samples after transduction with undiluted cell-free 293T supernatant as a function of time after termination of incubation with virus supernatant.
  • FIG. 12 shows the intensity of the EGFP fluorescence signal over a period of 1 to 24 hours after transduction with various EGFP-expressing or EGFP-labeled PFV vector particles.
  • PFV vectors were engrafted by transient transfection of 293T cells
  • pcoPG4 (p71, authentic full-length PFV p71 gag); pcoPG4
  • CEGFP (p71 EG, full-length PFV p71 gag with C-terminal EGFP fusion); pcoPG4 1 -621 CEGFP (p68 EG, C-terminal truncated PFV p68Gag with C-terminal EGFP fusion); Absence of gag
  • Transfer vector pcziEGqP (- / CMV EG) transfected.
  • HT1080 target cells with undiluted virus vector supernatant were transduced by spinoculation (30 min, 10 ° C, 1200g) at various time points after sowing in the presence (dashed lines) or absence (solid lines) of 100 g / ml cycloheximide and EGFP transgene expression of different samples harvested at the same time, measured by flow cytometry.
  • As control HT1080 target cells were used, which were not incubated with cell-free 293T supernatant (- / - (mock)).
  • A cycloheximide structure and information on the mode of action.
  • B cycloheximide structure and information on the mode of action.
  • E protein transfer. Mean EGFP fluorescence intensity (EGFP MFI) of the different target cell samples of EGFP-labeled PFV particles after transduction with undiluted cell-free 293T supernatant in
  • PFV vectors were generated by transiently transfecting 293T cells using an expression-optimized 4-component PFV vector system according to WO-A-2010/11608 using different amounts of the transfer vectors pcziEGgP (CMV) and pSFFVU3 EGqP (SFFV) of the present invention produced as described in FIG. 3. For all samples, the same amounts of packaging plasmids were used and the total amount of DNA used
  • RNA was extracted from the transfected 293T cells and the copy number of EGFP mRNA in the individual samples was determined using an EGFP-specific quantitative PCR (cell RNA, dashed lines).
  • Cell RNA levels were normalized to the amount of total RNA isolated.
  • viral particles were pelleted from 10 ml supernatant by ultracentrifugation and then the protein composition was analyzed by Western blot and the EGFP mRNA packaging (viral RNA, solid lines) with an EGFP-specific quantitative PCR.
  • Virus RNA levels were normalized to the amount of detectable capsid (Gag) protein.
  • HT1080 target cells were transduced with 1 ml samples of the various undiluted vector supernatants and EGFP transgene expression (MFI, dotted lines) was measured 48 hours later by flow cytometry.
  • Example 1 In order to determine the necessity of the foamyviral enzymatic functions for the stable genetic modification of target cells by means of foamyviral vectors, various EGFP transgene-containing FV vector particles were produced with a standard PFV transfer vector (pMD9, Figure 4A). These differed in the type of FV-Pol packaging plasmid used. Particles were made containing a wild type pol protein (wt), or pol protein variants, which included either an enzymatically inactive domain of the protease (iPR), reverse transcriptase (iR) or integrase (ilN).
  • wt wild type pol protein
  • iPR protease
  • iR reverse transcriptase
  • integrase integrase
  • the single spliced mRNA initiating in the FV 5 'LTR still has large 5' UT regions, so that also it should not allow transgene expression (FIG. 4A). Only the internal promoter-initiating, unspliced mRNA of the transgene cassette has good translational potential (FIG. 4A). This suggests that FV vector particles, in addition to the viral genomic RNA, also pack significant amounts of subgenomic mRNAs that primarily contribute to the transient transgene expression of vectors that do not reverse
  • FV vector particles to which only transgene-containing RNAs without the essential viral cis-active sequences (CASI and CASII) are offered in the packaging cell, can package them and lead to their translation into target cells.
  • FV packaging plasmids for Gag, Pol and Env expression vectors were cotransfected during vector production, which were either only those contained in the standard PFV vector pMD9
  • Transgene expression cassette with an additional polyadenylation signal contained (pSFFV U3 EGFP) or instead of the 3 'LTR sequences of the pMD9 vector (pSSFV U3 EGFP PFV 3') had ( Figure 4A).
  • the content of EGFP gene-containing RNA in the viral particles was almost identical ( Figure 4C). This finding suggests that PFV particles can efficiently package mRNAs that do not contain authentic PFV sequences and transduce them into their target cells after transduction.
  • Example 2 It is known that splicing mRNAs with the binding of snRNAs and the spliceosome complex can stabilize them and tag them for nuclear export. To analyze the influence of the transcription cassette on the FV capsid-mediated RNA transfer, equal amounts of different EGFP transgene-containing expression vectors with PFV Gag, Pol and Env
  • Packaging plasmids are cotransfected for vector particle production ( Figure 5A). After transduction of target cells with different dilutions vector containing
  • Transgene expression can be measured.
  • Example 3 it was analyzed whether other retroviral vector systems are also capable of RNA transfer independent of viral sequences on the transfer vector and compared their efficiency with the FV vector systems.
  • HIV-1, MLV and PFV vector particles were cotransfected by a CMV promoter-intron-driven transfer vector with the respective
  • Packaging plasmids are cotransfected and the transgene expression in target cells is determined by flow cytometry after transduction with undiluted vector supernatants (FIG. 7). For comparison, analogous vector particles were used in parallel with the
  • Results show that, in absolute terms, PFV-mediated RNA transfer is at least 3-fold more potent than HIV-1 mediated, whereas only very poor MLV-mediated RNA transfer was measured (Figure 7A). If the titers of the analogous vector supernatants are taken into consideration for stable transgene expression as a measure of the physical particle amount, then the RNA transfer efficiency per vector particle in PFV vectors is approximately 50 times higher in comparison to that of HIV-1 (FIG. 7B).
  • Transcriptase and integrase indispensable (FIG. 8A).
  • a CMV promoter-intron driven transfer vector was cotransfected with various combinations of packaging plasmids. The potential of the so generated
  • transgene expression was at least 150-fold lower than in the
  • FV transfer vectors were prepared for stable or transient expression of a humanized form of Cre recombinase, producing corresponding viral vector supernatants and determining the enzymatic function in target cells (Figure 9A).
  • Embryonic mouse fibroblasts were transduced as target cells containing a LoxP flanked EYFP expression cassette in the ROSA26 locus, which becomes transcriptionally active only after Cre-mediated recombination (Srinivas 2001) ( Figure 9).
  • Example 6 In order to examine the influence of the transcription cassette as well as the enzyme activities of the Pol protein on the transient gene transfer and the time course of the
  • PFV vector supernatants were screened for transgene expression by transient transfection of 293T cells using a
  • the transfer vectors puc2MD9 EGqP (MD9), pSFFVU3 EGqP (SFFV EGqP) and pcziEGqP (CMV EGqP) (FIG. 4A or FIG. 5) were used.
  • the vector pcziEGqP is a CMV promoter-intron-driven transfer vector, while the vector pSFFVU3 EGqP has an intronless SFFVU3 promoter.
  • the vector particles containing the standard PFV transfer vector puc2MD9 additionally differed in the type of cotransfected Pol packaging construct:
  • the construct pcoPP2 In the wild-type construct pcoPP all enzymatic activities are intact, the construct pcoPP2 (iRT) has an inactivated reverse transcriptase, the construct pcoPP3 (ilN) an inactivated integrase.
  • the produced vector supernatants were used to transduce HT1080 target cells by spinoculation (30 min, 10 ° C, 1200 g). Transduction occurred at different time points after seeding, EGFP transgene expression was measured by flow cytometry (Figure 10A). The result of flow cytometry revealed that a transduction of the target cells with virus particles obtained by transfection of 293T cells with the standard PFV-
  • Figure 11A Measurement of expression over a longer observation period (Figure 11A) revealed that only transduction with vector particles produced with the standard PFV transfer vector puc2MD9 on co-transfection with the wild-type Pol packaging construct (MD9 / wt) stable expression over the entire observation period of 16 days (FIG. 11 B).
  • Example 7 In a further experiment, the EGFP fluorescence signal was measured in the target cells after transduction with various EGFP-expressing or EGFP-labeled PFV vector particles over a period of 1 to 24 hours after infection.
  • the PFV vector supernatants were prepared by transiently transfecting 293T cells using an expression-optimized 4-component PFV vector system (see Example 1). The were
  • Transfer vectors puc2MD9 EGqP (MD EG), pcziEGqP (CMV EG) as well as the empty vector pczi (CMV) used.
  • the particles produced with the transfer vector pcziEGqP (CMV EG) also differed in the type of cotransfected gag packaging construct.
  • the constructs pcoPG4 (p71,
  • pcoPG4 CEGFP p71 EG, full-length PFV p71 gag with C-terminal EGFP fusion
  • pcoPG4 1 -621 CEGFP p68 EG, C-terminal truncated PFV p68Gag with C-terminal EGFP fusion
  • no gag packaging construct is cotransfected.
  • the produced vector supernatants were used to transduce HT1080 target cells by spinoculation (30 min, 10 ° C, 1200 g). used. The transfection took place at different times after the
  • the measured fluorescence is thus indicative of EGFP expression
  • Protein transfer was determined by measuring the EGFP fluorescence intensity of target cells transduced with EGFP capsid-labeled PFV vector particles ( Figure 12E, solid lines). The measurement revealed that vector particles resulting from cotransfection with a terminally truncated PFV p68Gag (p68 EG) show stronger target cell fluorescence as a result of protein transfer than vector particles found in co-transfection with a full-length PFV p71 gag (FIG. p71 EC). Furthermore, it was shown that the fluorescence produced by transduction with EGFP-labeled, capsidless
  • Vector particles are triggered in which no gag packaging construct
  • CMV EG cotransfected cotransfected
  • fluorescence in the cells transfected with EGFP-tagged PFV vector particles depends on the transfer of the EGFP-labeled particles, rather than on the transfer and subsequent expression of the RNA in the target cell, addition of cycloheximide to the infection approaches in this case no influence on the measured fluorescence intensity (FIG. 12E, dashed lines).
  • Example 8 To detect the dose-dependency of RNA transfer, first, PFV vectors were prepared by transiently transfecting 293T cells using an expression-optimized 4-component PFV vector system as described in Example 1. There were different amounts of
  • Transfer vectors pcziEGgP (CMV) and pSFFVU3 EGqP (SFFV) were used. After 48 hours, the cell-free viral supernatants were harvested and 1 ml each of the various undiluted vector supernatants were used to transduce HT1080 target cells with the vector particles contained in the supernatant. Quantitative PCR was used to determine the amount of cell RNA after extraction of the total RNA from the transfected 293T cells and the amount of virus RNA after extraction of the RNA from the viral particles pelleted by ultracentrifugation of the supernatant (10 ml). EGFP transgene expression in the target cells was measured 48 hours after infection by flow cytometry. It showed that a proportionality between the transfection of the 293T cells

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Abstract

L'invention concerne un système de vecteur rétroviral comportant un vecteur de transfert, contenant au moins une cassette d'expression destinée à contenir une séquence de nucléotides codant pour un produit de gène hétérologue par rapport au rétrovirus concerné, en particulier non rétroviral, qui est caractérisé en ce qu'il ne contient aucune séquence du rétrovirus concerné. D'autres objets de la présente invention concernent des cellules hôtes qui sont transfectées avec le système de vecteur, des procédés pour la préparation des cellules hôtes, un kit pour l'expression d'un produit de gène hétérologue par rapport au rétrovirus concerné, en particulier non rétroviral, qui contient le système de vecteur, ainsi qu'un procédé pour l'expression d'un produit de gène hétérologue par rapport au rétrovirus concerné, en particulier non rétroviral, dans une cellule à l'aide du système de vecteur.
PCT/EP2012/058057 2011-05-09 2012-05-02 Système de vecteur rétroviral minimal WO2012152632A1 (fr)

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WO2015028683A1 (fr) * 2013-09-02 2015-03-05 Cellectis Procédé à base d'arn pour obtenir des vecteurs rétroviraux intégrés de manière stable
EP3822346A1 (fr) 2019-11-14 2021-05-19 Technische Universität Dresden Utilisation de lignées de cellules d'emballage par génie génétique pour la production de particules de virus recombinants
EP4286401A1 (fr) 2022-06-03 2023-12-06 Technische Universität Dresden Utilisation d'une protéine artificielle ou d'un acide nucléique codant la protéine artificielle pour fournir un complexe de ribonucléoprotéines fonctionnel

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015028683A1 (fr) * 2013-09-02 2015-03-05 Cellectis Procédé à base d'arn pour obtenir des vecteurs rétroviraux intégrés de manière stable
US10378026B2 (en) 2013-09-02 2019-08-13 Cellectis RNA based method to obtain stably integrated retroviral vectors
EP3822346A1 (fr) 2019-11-14 2021-05-19 Technische Universität Dresden Utilisation de lignées de cellules d'emballage par génie génétique pour la production de particules de virus recombinants
EP4286401A1 (fr) 2022-06-03 2023-12-06 Technische Universität Dresden Utilisation d'une protéine artificielle ou d'un acide nucléique codant la protéine artificielle pour fournir un complexe de ribonucléoprotéines fonctionnel

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