WO2012152632A1 - Minimal retrovirus vector system - Google Patents
Minimal retrovirus vector system Download PDFInfo
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- 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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Classifications
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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
The present invention relates to a retrovirus vector system having a transfer vector, comprising at least one expression cassette for accommodating a nucleotide sequence which codes for a gene product, more particularly a non-retroviral gene product, which is heterologous in relation to the retrovirus in question, the transfer vector being characterized in that it does not contain any sequences of the retrovirus in question. The invention also relates to host cells transfected with the vector system, methods for producing the host cells, a kit for expressing a gene product, more particularly a non-retroviral gene product, which comprises the vector system and which is heterologous in relation to the retrovirus in question, and also a method for expressing a gene product, more particularly a non-retroviral gene product, which is heterologous in relation to the retrovirus in question, in a cell, using the vector system.
Description
Minimales Retrovirus-Vektorsystem Minimal retrovirus vector system
Die vorliegende Erfindung betrifft ein Retrovirus-Vektorsystem mit einem The present invention relates to a retrovirus vector system having a
Transfervektor, enthaltend mindestens eine Expressionskassette zur Aufnahme einer für ein in Bezug auf das betreffende Retrovirus heterologes, insbesondere nicht- retrovirales Genprodukt codierenden Nukleotidsequenz, der dadurch gekennzeichnet ist, dass er keine Sequenzen des betreffenden Retrovirus enthält. Weitere 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. Further
Gegenstände der vorliegenden Erfindung betreffen Wirtszellen, die mit dem Objects of the present invention relate to host cells associated with the
Vektorsystem transfiziert sind, Verfahren zur Herstellung der Wirtszellen, ein Kit zur Expression eines in den Bezug auf das betreffende Retrovirus heterologen, insbesondere nicht-retroviralen Genprodukts, dass das Vektorsystem enthält, sowie ein Verfahren zur Expression eines in Bezug auf das betreffende Retrovirus heterologen, insbesondere nicht-retroviralen Genprodukts in einer Zelle unter Verwendung des Vektorsystems. 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.
Retroviren verpacken zwei Kopien ihrer RNA-Genome (+ -strängige Orientierung, einzelsträngig, daher die gleiche Struktur wie zelluläre mRNA) während der Retroviruses pack two copies of their RNA genomes (+ stranded orientation, single-stranded, therefore the same structure as cellular mRNA) during the
Virusgenerierung in das virale Kapsid. Während ihres Replikationszyklus werden diese verpackten RNA-Genome durch die retrovirale Reverse Transkriptase (RT) in eine Einzelkopie doppelsträngiger (ds) DNA umgewandelt und nachfolgend in das Wirtszellgenom durch einen Prozess integriert, der vom viralen Integrase- (IN) Protein abhängig ist. Der Zeitpunkt der reversen Transkription ist bei den Virus 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. The time of reverse transcription is at
verschiedenen Unterfamilien der Retroviren verschieden (siehe Fig. 1 ). Die different subfamilies of retroviruses different (see Fig. 1). The
Orthoretroviren (z.B. murines Leukämievirus (MLV), humanes Immundefizienzvirus (HIV)), revers-transkribieren ihr Genom nach dem Eintritt in die Zielzelle. Orthotrophic viruses (e.g., murine leukemia virus (MLV), human immunodeficiency virus (HIV)) reverse transcribe their genome upon entry into the target cell.
Demgegenüber revers-transkribieren Spumaretroviren (Foamy Viren (FV)) ihr verpacktes RNA-Genom bereits meist während des Virusaufbaus in den virusproduzierenden Zellen. FV-Partikel, die virale dsDNA-Genome mit vollständiger Länge enthalten, sind für die Mehrheit von Infektionsereignissen verantwortlich.
Die Integration retroviraler Genome in das Wirtszellgenom führt zu einer Langzeitexpression in den infizierten Zellen. Sofern keine Integration erfolgt, sind zwei verschiedene Arten der transienten retroviralen Proteinexpression bekannt (Fig. 2). Zum einen, falls weiter eine reverse Transkription stattfindet, jedoch die von der retroviralen Integrase vermittelte Integration verhindert wird, wird eine im wesentlichen transiente Transkription, ausgehend von nicht-integrierten, episomalen viralen DNA-Genomen, beobachtet. Diese episomalen viralen DNA-Genome können mit sehr geringer Häufigkeit in das Wirtszellgenom durch nicht-homologe In contrast, spumetroviruses (Foamy viruses (FV)) already reverse-transcribe their packaged RNA genome during virus building in the virus-producing cells. 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
Rekombinationsereignisse (ähnlich wie bei einer stabilen Plasmidtransfektion) integrieren, und führen zu einer Langzeitexpression in diesen Zellen. Zum zweiten können, falls keine reverse Transkription stattfindet, die zwei Kopien der verpackten viralen RNA-Genome translatiert werden, wenn sie im Zytoplasma freigesetzt werden. Im Allgemeinen führt der ersten Mechanismus zu einer längeren und stärkeren Proteintranslation als der zweite Mechanismus. 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.
Mehrere Forschungsgruppen verwendeten diese Phänomene zur Entwicklung retroviraler Gentransfersysteme zur transienten Transgenexpression (Fig. 2). So sind beispielsweise die sogenannten integrationsdefizienten retroviralen Vektoren (RV) (ΔΙΝ-Systeme) bekannt, die eine enzymatisch inaktivierte retrovirale Integrase enthalten (Banasik et al. (2010) Gene Ther. 17:150-157). Die durch einen derartigen RV hervorgerufene Transgenexpression nimmt stetig über eine Zeitspanne von 10- 14 Tagen in sich teilenden Zellen ab, kann jedoch auch stabil über Wochen in ruhenden Zellen sein. Allerdings kann selbst in sich schnell teilenden Zellen eine geringe Langzeittransgenexpression aufgrund nicht-viral vermittelter nicht-homologer Rekombination von viralen und zellulären DNA-Genomen nachgewiesen werden, die zu einer stabilen Integration des viralen Genoms in das Wirtszellgenom führt. Several research groups have used these phenomena to develop retroviral gene delivery systems for transient transgene expression (Figure 2). Thus, for example, the so-called integration-deficient retroviral vectors (RV) (ΔΙΝ systems) are known which contain an enzymatically inactivated retroviral integrase (Banasik et al. (2010) Gene Ther. 17: 150-157). 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. However, even in rapidly dividing 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.
Derartige Systeme sind auch kürzlich für auf Foamy Viren basierenden Vektoren beschrieben worden (Deyle et al. (2010) J. Virol. 84:9341 -9349). Such systems have also recently been described for foamy virus-based vectors (Deyle et al., (2010) J. Virol. 84: 9341-9349).
Darüber hinaus wurden transiente RV-Systeme entwickelt, bei denen ein In addition, transient RV systems have been developed in which a
sogenannter Partikel-vermittelnder mRNA-Transfer (PMT) stattfindet, in welchen die Umwandlung des verpackten RNA-Genoms in DNA durch katalytische Inaktivierung der Reversen-Transkriptase-Domäne des retroviralen Pol-Proteins oder durch
Modifikation essentieller cis-wirkender viraler Sequenzen verhindert wird (Fig. 2) (Galla et al. (2008) J. Virol. 82:3069-3077; Galla et al. (2004) Mol. Cell 16:309-315). Derartige RV ermöglichen eine transiente Transgentranslation in transduzierten Zielzellen direkt von der eingeschleusten viralen RNA, die die gleiche Struktur und Orientierung wie bei einer zellulären mRNA aufweist. Im Gegensatz zu ΔΙΝ-so-called particle-mediating mRNA transfer (PMT) takes place in which the conversion of the packaged RNA genome into DNA by catalytic inactivation of the reverse transcriptase domain of the retroviral Pol protein or by Modification of essential cis-acting viral sequences is prevented (Figure 2) (Galla et al., (2008) J. Virol., 82: 3069-3077; Galla et al. (2004) Mol. Cell 16: 309-315). Such RVs allow transient transgene translation into transduced target cells directly from the introduced viral RNA having the same structure and orientation as a cellular mRNA. Unlike ΔΙΝ-
Systemen ist die PMT-vermittelte Transgenexpression vollständig transient, und es wurden keine nicht-homologen Rekombinationsereignisse von viralen und zellulären Nukleinsäuren beobachtet, die in einer genetischen Langzeitmodifikation resultieren könnten. Die Höhe und Dauer der PMT-vermittelten Transgenexpression ist deutlich geringer und kürzer als diejenige durch ΔΙΝ-RV-Systeme. Der Grund hierfür ist, dass die PMT-vermittelte Transgenexpression von den zwei Kopien des viralen RNA- Genoms pro Viruspartikel, die in das Zytoplasma der Zielzelle abgegeben werden, ausgeht, während die episomale Abgabe einer Kopie des revers transkribierten viralen DNA-Genoms pro Viruspartikel bei ΔΙΝ-RV-Systemen zur Erzeugung mehrfacher mRNA-Kopien aufgrund der de novo Transgentranskription durch die zelluläre Transkriptionsmaschinerie und daher zur Amplifikation der In systems, 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 ΔΙΝ-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 ΔΙΝ-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
Transgenexpression führt. Transgene expression leads.
Beiden Systemen gemeinsam ist, dass sie sogenannte cis-wirksame virale Common to both systems is that they are called cis-acting viral
Sequenzen zur spezifischen Verpackung von RNA in das retrovirale Partikel benötigen (Fig. 2). Es wird angenommen, dass Retrovieren selektiv ihr Volllängen- RNA-Genom durch Erkennung spezifischer Verpackungssequenzen (RNA-Stamm- Schleife-Strukturen) verpacken. Es ist jedoch eine Verpackung zellulärer RNA- Sequenzen in Anwesenheit und Abwesenheit verpackter viraler RNA-Sequenzen bekannt (Piver et al. (2006) J. Virol. 80:9889-9895). Need sequences for specific packaging of RNA in the retroviral particle (Fig. 2). It is believed that retrovars selectively package their full-length RNA genome by recognizing specific packaging sequences (RNA stem-loop structures). However, packaging of cellular RNA sequences in the presence and absence of packaged viral RNA sequences is known (Piver et al., (2006) J. Virol., 80: 9889-9895).
WO-A-2010/1 1 1608 beschreibt ein Foamy Virus-Vektorsystem, in dem mindestens ein rekombinanter Vektor eine Expressionssequenz umfasst, die mindestens eine Komponente eines Foamy-Virus-Partikels codiert, wobei mindestens ein Codon der Expressionssequenz zur Expression in Homo sapiens optimiert ist. 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.
Es besteht weiter Bedarf an der Verbesserung retroviraler Expressionssysteme hinsichtlich unerwünschter Einschleusungen retroviraler Sequenzen in das There remains a need for the enhancement of retroviral expression systems for unwanted infiltrations of retroviral sequences into the
Wirtsgenom.
Zur Lösung dieser Aufgabe wird erfindungsgemäß ein Retrovirus-Vektorsystem bereitgestellt, das (a) einen oder mehrere rekombinante Vektor(en), der/die Host genome. To achieve this object, a retroviral vector system is provided according to the invention, which (a) one or more recombinant vector (s), the /
Expressionssequenzen enthält/enthalten, welche mindestens für die zur Herstellung verpackungsfähiger Viruspartikel essentiellen Proteine des Retrovirus codieren, und (b) einen Transfervektor umfasst, der mindestens eine Expressionskassette zur Aufnahme einer für ein in Bezug auf das Retrovirus heterologes Genprodukt, vorzugsweise ein nicht-retrovirales Genprodukt codierenden Nukleotidsequenz enthält, wobei der Transfervektor keine Sequenzen des betreffenden Retrovirus aufweist. Contains expression sequences which encode at least the proteins of the retrovirus essential for the preparation of packageable virus particles, and (b) 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.
Des Weiteren wurde erfindungsgemäß festgestellt, dass es für eine retroviral vermittelte transiente Expression eines Genprodukts in Zielzellen ausreichend ist, in Verpackungszellen Gag- und Env-Proteine bereitzustellen, die in der Furthermore, it has been found according to the invention that, for retrovirally mediated transient expression of a gene product in target cells, it is sufficient to provide gag and Env proteins in packaging cells which are known in the art
Verpackungzellinie über einen geeigneten Transfervektor bereitgestellte, Packaging cell line provided via a suitable transfer vector,
verpackbare RNA verpacken und so zur Transduktion geeigneter Zielzellen führen, in denen das eingeführte genetische Material zu einer transienten Expression des Genprodukts führt. Daher stellt die vorliegende Erfindung auch allgemein ein minimales retrovirales Vektorsystem bereit, dass (1 ) einen oder mehrere packaging packageable RNA and thus lead to the transduction of suitable target cells in which the introduced genetic material leads to a transient expression of the gene product. Therefore, the present invention also generally provides a minimal retroviral vector system in that (1) one or more
rekombinante Vektor(en), der/die in Bezug auf den betreffenden Virus nur solche Expressionssequenzen enthält/enthalten, die für die Proteine Env und Gag des Retrovirus codieren und (2) einen (beliebigen) Transfervektor umfasst, der mindestens eine Expressionskassette zur Aufnahme einer für ein Genprodukt codierenden Nukleotidsequenz enthält. Unter diesem Aspekt der vorliegenden Erfindung kann der Transfervektor auch retrovirale Sequenzen, wie bspw. c/s-aktive Verpackungssequenzen enthalten. Recombinant vector (s) 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. In this aspect of the present invention, the transfer vector may also contain retroviral sequences, such as c / s-active packaging sequences.
Geeignete Retrovirus-Vektorsysteme gemäß der vorliegenden Erfindung sind bspw. HIV-1 -, MLV- und Foamy-Virus-Vektorsysteme. Suitable retroviral vector systems according to the present invention are, for example, HIV-1, MLV and foamy virus vector systems.
Die Vektoren oder der Vektor gemäß Komponente (a) des erfindungsgemäßen Vektorsystems stellt bzw. stellen bei Expression in einer geeigneten The vectors or the vector according to component (a) of the vector system according to the invention represents or provide expression in a suitable
Verpackungszelllinie mindestens diejenigen Virus-Proteine bereit, die für die Packing cell line at least those virus proteins ready for the
Erzeugung und Ausschleusung verpackungsfähiger Virus-Partikel essentiell sind.
Wie bereits vorstehend dargestellt, sind dies üblicherweise mindestens die Proteine Gag und Env. Darüber hinaus können durch die Komponente (a) auch Pol und/oder weitere retrovirale Proteine, wie bspw. bei HIV-1 z.B. eines oder mehrere der Proteine Tat, Rev, Vpr und Vpu bereitgestellt werden. Generation and discharge of packageable virus particles are essential. As already indicated above, these are usually at least the proteins Gag and Env. In addition, by the component (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.
Eine besonders bevorzugte Ausführungsform der vorliegenden Erfindung ist ein Foamy-Virus-Vektorsystem, das (a1 ) einen rekombinanten Vektor, der eine A particularly preferred embodiment of the present invention is a foamy virus vector system comprising (a1) a recombinant vector containing a
Expressionssequenz enthält, die für ein Gag Protein eines Foamy Virus codiert, und einen rekombinanten Vektor, der eine Expressionssequenz enthält, die für ein Env Protein eines Foamy Virus codiert; oder (a2) einen rekombinanten Vektor, der eine Expressionssequenz enthält, die für ein Gag Protein eines Foamy Virus codiert, und eine Expressionssequenz enthält, die für ein Env Protein eines Foamy Virus codiert; und (b) einen Transfervektor umfasst, der mindestens eine Expressionskassette zur Aufnahme einer für ein Nicht-Foamy-Virus-Genprodukt codierenden Containing an expression sequence coding for a gag protein of a foamy virus, and a recombinant vector containing an expression sequence coding for a Env protein of a foamy virus; or (a2) a recombinant vector containing an expression sequence encoding a gag protein of a foamy virus and containing an expression sequence encoding a Env protein of a foamy virus; and (b) a transfer vector comprising at least one expression cassette for receiving a non-foamy virus gene product
Nukleotidsequenz enthält. Contains nucleotide sequence.
Die vorliegende Erfindung beruht auf der überraschenden Erkenntnis, dass - entgegen der Lehre des Standes der Technik - in einem retroviralen Vektorsystem wie beispielsweise HIV-1 -, MLV- oder Foamy-Virus-Vektorsystemen 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
Transfervektoren eingesetzt werden können, die keinerlei von dem betreffenden Retrovirus wie einem Foamy-Virus abgeleitete Nukleotidsequenzen, insbesondere keine c/s-wirksamen Sequenzen, wie bspw. im Fall des Foamy Virus CAS I / CAS II, enthalten und zu einer ausschließlich transienten Expression eines Transgens in entsprechenden Zielzellen verwendet werden können. 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.
Gemäß der Erfindung ist somit ein Transfervektor, der keine Sequenzen des betreffenden Retrovirus enthält, insbesondere ein Transfervektor, dessen According to the invention, a transfer vector which does not contain sequences of the relevant retrovirus, in particular a transfer vector whose
Nukleotidsequenz derart ausgebildet ist, dass sie in einer Sequenz-Fenstergröße von etwa 100 oder mehr Nukleotiden eine Homologie zu einer Nukleotidsequenz des betreffenden Retrovirus, vorzugsweise eines beliebigen Retrovirus, von höchstens 50%, vorzugsweise höchstens 40 %, mehr bevorzugt höchstens 10 % oder weniger aufweist.
Im Rahmen der vorliegenden Erfindung wurde, wie bereits vorstehend erwähnt, festgestellt, dass für ein erfindungsgemäß bevorzugtes funktionales Vektorsystem, basierend auf einem Foamy Virus, insbesondere prototypischen FV (PV; auch bekannt als humaner FV (HFV)), für eine effiziente Verpackung lediglich die Proteine Gag und Env über zwei rekombinante Vektoren (Komponente (a1 ) des 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 , In the context of the present invention, as already mentioned above, it has been found that for a preferred functional vector system based on a foamy virus, in particular prototypical FV (PV; also known as human FV (HFV)), for efficient packaging only the Proteins Gag and Env via two recombinant vectors (component (a1) of the
erfindungsgemäßen Foamy-Virus-Vektorsystems) oder einen rekombinanten Vektor (Komponente (a2) des Foamy-Virus-Vektorsystems der vorliegenden Erfindung), der/die entsprechende Expressionssequenzen enthält/enthalten, zur Verfügung gestellt werden müssen, um infektiöse Partikel, die über den erfindungsgemäßen Transfer-Vektor bereitgestellte RNA enthält, herzustellen. In bestimmten 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. In particular
Ausführungsformen der vorliegenden Erfindung kann es jedoch vorteilhaft sein, eine Expressionssequenz für ein Pol-Protein eines Foamy-Virus bereitzustellen. Eine derartige Pol-Expressionssequenz kann auf einem oder beiden der Vektoren gemäß Komponente (a1 ) und/oder auf dem Vektor gemäß Komponente (a2) bereitgestellt werden. In einer anderen Ausführungsform der Erfindung kann jedoch auch ein weiterer rekombinanter Vektor (c) bereitgestellt werden, der eine However, embodiments of the present invention may be advantageous in providing an expression sequence for a polymorphic protein of a foamy virus. Such 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). In another embodiment of the invention, however, a further recombinant vector (c) may be provided which comprises a
Expressionssequenz enthält, die für ein Pol-Protein des Foamy-Virus codiert. In einer weiteren vorteilhaften Variante dieser Ausführungsform der Erfindung ist die jeweilige Expressionssequenz für das Pol-Protein genetisch biosynthetisch inaktiviert. Contains expression sequence coding for a Pol protein of foamy virus. In a further advantageous variant of this embodiment of the invention, the respective expression sequence for the pol protein is genetically biosynthetically inactivated.
Weiterhin wird erfindungsgemäß ins Auge gefasst, einen oder beide der Furthermore, the invention envisages one or both of the
rekombinanten Vektoren gemäß (a1 ) oder den rekombinanten Vektor gemäß (a2) und/oder gegebenenfalls den rekombinanten Vektor gemäß (c) mit einer recombinant vectors according to (a1) or the recombinant vector according to (a2) and / or optionally the recombinant vector according to (c) with a
Expressionssequenz auszustatten, die für eine nicht-funktionale Foamy-Virus- Integrase und/oder eine nicht-funktionale Foamy-Virus-Reverse-Transkriptase und /oder eine nicht-funktionale Foamy-Virus-Protease codiert. An expression sequence coding for a non-functional foamy virus integrase and / or a non-functional foamy virus reverse transcriptase and / or a non-functional foamy virus protease.
Das Foamy-Virus-Vektorsystem der vorliegenden Erfindung ist vorzugsweise von einem humanen Foamy Virus (HFV oder auch PFV, s.o.) abgeleitet, kann jedoch auch von aus Affen isolierten Foamy Viren (SFV) oder anderen Säugetieren isolierten Foamy Viren oder Mischformen abgeleitet sein bzw. auf solchen basieren. 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.
Erfindungsgemäß ist es weiter bevorzugt, mindestens eine der According to the invention it is further preferred that at least one of
Expressionssequenzen (also z.B. im Fall des Foamy-Virus-Vektorsystems Expression sequences (i.e., in the case of the foamy virus vector system
insbesondere Gag und/oder Env und/oder Pol) zur Expression in Homo sapiens zu
optimieren. In Bezug auf das Foamy-Virus-Vektorsystem wird in diesem Zusammenhang ausdrücklich auf die WO-A-2010/1 161 1 1608 und deren in particular Gag and / or Env and / or Pol) for expression in Homo sapiens too optimize. With respect to the foamy virus vector system is in this context expressly to WO-A-2010/1 161 1 1608 and their
diesbezüglichen Offenbarungsgehalt verwiesen. Besonders bevorzugte Transfervektoren der vorliegenden Erfindung weisen eine Nukleotidsequenz auf, die aus der Gruppe, bestehend aus SEQ ID NO:1 und SEQ ID NO: 2, ausgewählt ist. Der Vektor gemäß SEQ ID NO: 1 wird auch als Vektor pczi bezeichnet und wurde auf Basis des pcDNA3.1 +zeo Vektors (Invitrogen) und des CMV Introns A der pHIT Vektoren (Soneoka ei al. (1995) Nucleic Acids Res. 23(4), 628-633) hergestellt (siehe Heinkelein ei al. (2002) J. Viral. 76(9), 3774-3783). Der Vektor gemäß SEQ ID NO: 2 wird auch als Vektor pSFFVU3 bezeichnet und basiert auf dem pEGFP-Vektor (Clontech), bei dem der CMV Promoter durch eine SFFV U3 Promotor-Variante ersetzt wurde, bei der eine 42 Bp-Duplikation referenced disclosure. 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
(GCAGTTTCGGCCCCGGCCCGGGGCCAAGAACAGATGGTCACC; SEQ ID NO: 3) des vollständigen SFFV U3 Promotors fehlt. Weitere Merkmale der Vektoren gemäß SEQ ID NO: 1 und 2 sind dem beigefügten Sequenzprotokoll zu entnehmen. (GCAGTTTCGGCCCCGGCCCGGGGCCAAGAACAGATGGTCACC; SEQ ID NO: 3) of the complete SFFV U3 promoter is missing. Further features of the vectors according to SEQ ID NO: 1 and 2 can be found in the attached sequence listing.
Ein„Genprodukt" gemäß der vorliegenden Erfindung kann jegliches Produkt sein, das über eine Expressionskassette in einem in der vorliegenden Offenbarung definierten Transfervektor in geeigneten Zielzellen exprimierbar ist. Dabei umfasst erfindungsgemäß der Ausdruck„Genprodukt" unmittelbar aus der Transkription hervorgehende Expressionsprodukte als auch auf von dem Transkriptionsprodukt abgeleitete Expressionsprodukte wie insbesondere Translationsprodukte oder Produkte, die aus einer Prozessierung des unmittelbaren Genprodukts hervorgehen. Eine in einer Expressionskassette eines Transfervektors der vorliegenden Erfindung eingefügte Nukleotidsequenz kann in einer Ausführungsform für Peptide oder Proteine, insbesondere nicht-retroviralen Ursprungs, insbesondere in Bezug auf den jeweiligen Retrovirus heterologe Peptide und/oder Proteine (z.B. ein Nicht-Foamy- Virus-Protein) codieren. Der Fachmann kennt selbstverständlich weitere 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. In one embodiment, 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. Of course, the person skilled in the art knows more
Transkriptionsprodukte, die in Zellen nicht in Peptide/Proteine translatiert werden. Beispiele hierfür sind tRNAs. Weitere nicht-peptidische bzw. nicht-proteinartige Genprodukte sind Antisense-RNAs, siRNAs, shRNAs, miRNAs (auch als microRNAs bekannt), pre-miRNAs und pri-miRNAs.
Bevorzugt ist in dem Transfervektor der erfindungsgemäßen Vektorsysteme in die Expressionskassette funktional eine Nukleotidsequenz eingefügt, die für mindestens ein heterologes, insbesondere nicht-retrovirales Protein codiert. Die 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. In the transfer vector of the vector systems according to the invention, a nucleotide sequence which codes for at least one heterologous, in particular non-retroviral protein is preferably inserted into the expression cassette. The
Nukleotidsequenz kann ebenfalls mindestens teilweise für ein Reporterprotein oder - peptid codieren. Geeignete Reporterproteine bzw. -peptide sind im Stand der Nucleotide sequence may also at least partially encode a reporter protein or peptide. Suitable reporter proteins or peptides are in the state of
Technik bekannt. Beispielsweise kann das Reporterprotein ein GFP-Protein oder eine Derivat davon wie beispielsweise EGFP und ähnliche Fluoreszenz generierende Proteine sein. Der Begriff„Expressionssequenz" bedeutet erfindungsgemäß eine Technique known. For example, the reporter protein may be a GFP protein or a derivative thereof such as EGFP and similar fluorescence-generating proteins. The term "expression sequence" means according to the invention a
Nukleotidsequenz, in welcher eine Nukleotidsequenz, die für ein Protein, Nucleotide sequence in which a nucleotide sequence coding for a protein,
beispielsweise eines Foamy-Virus-Proteins wie Gag, Env bzw. Pol codiert, vorliegt und gegebenenfalls operabel mit geeigneten Promotor und Terminator-Sequenzen verbunden ist, so dass das Protein in einer geeigneten Wirtszelle exprimiert wird. Weitere funktionale Sequenzen, die mit der Expressionssequenz verbunden sein können, sind Enhancersequenzen, IRES-Sequenzen und andere einem Fachmann bekannte funktionale Elemente, die zur Steuerung der Expression eines Proteins in einer Wirtszelle geeignet sind. Selbstverständlich stellt auch ein erfindungsgemäßer Transfervektor, in dem eine für mindestens ein Genprodukt codierende for example, a foamy virus protein such as Gag, Env or Pol, and optionally operably linked to 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. Of course, a transfer vector according to the invention, in which one coding for at least one gene product
Nukleotidsequenz in die Expressionskassette eingefügt ist, einen Vektor dar, der im Sinne der vorliegenden Erfindung eine entsprechende„Expressionssequenz" enthält. Somit gelten auch für den Transfervektor die obigen Ausführungen in Bezug auf mit der Expressionskassette (d.h. ohne eingefügte Nukleotidsequenz, die für mindestens ein Genprodukt codiert) bzw. der Expressionssequenz (d.h. mit eingefügter For the purposes of the present invention, 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
Nukleotidsequenz, die für mindestens ein Genprodukt codiert) operabel verbundener funktionaler Sequenzelemente entsprechend. Nucleotide sequence encoding at least one gene product) of operably linked functional sequence elements, respectively.
Die in dem Transfervektor gemäß der vorliegenden Erfindung vorhandene The present in the transfer vector according to the present invention
Expressionskassette ist somit eine Nukleotidsequenz, die wie vorstehend dargelegte expressionssteuerende Elemente enthält und vorzugsweise mit einer multiplen Klonierungsstelle (MCS) ausgestattet ist und/oder Sequenzen zur homologen 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
Rekombination zur Einfügung einer für das gewünschte Genprodukt, wie bspw. ein Protein, insbesondere ein nicht-retrovirales Protein, z.B. ein Nicht-Foamy-Virus- Protein, codierenden Nukleotisequenz enthält.
Ein weiterer Gegenstand der vorliegenden Anmeldung ist eine Wirtszelle, die mit einem Vektorsystem der vorliegenden Erfindung transfiziert ist. Geeignete Wirtszellen sind beispielsweise bakterielle Zellen wie E. coli, um das erfindungsgemäße Vektorsystem und/oder dessen Komponenten zu propagieren. Weitere bevorzugte Wirtszellen sind solche, die zur Herstellung und Ausschleusung von Retrovirus-Partikeln durch Expression mindestens der essentiellen Virus- Proteine über den/die Vektoren gemäß Bestandteil (a) bzw. Bestandteil (1 ) der Vektorsysteme der vorliegenden Erfindung ausgelegt sind. Im Fall eines bevorzugten retroviralen Systems wie einem erfindungsgemäßen Foamy-Virus-Systems sind die bevorzugten Wirtszellen daher z.B. solche, die zur Herstellung und Ausschleusung von Foamy-Virus-Partikeln durch Expression mindestens der Gag- und Env-Proteine eines Foamy Virus über die Vektoren gemäß (a1 ) beziehungsweise den Vektor gemäß (a2) ausgelegt sind. Beispiele solcher Zellen sind insbesondere Zelllinien wie humane 293 oder 293T-Zellen und Primaten-Zelllinien wie Cos1 - oder Cos7-Zellen. Recombination for insertion of a nucleotide sequence coding for the desired gene product, such as, for example, a protein, in particular a non-retroviral protein, for example a non-foamy virus protein. 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. In the case of a preferred retroviral system such as a foamy virus system according to the invention, therefore, 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.
Derartige Wirtszellen (auch als Verpackungszelllinien bezeichnet) dienen somit der Herstellung infektiöser Viruspartikel bzw. Virus-ähnlicher Partikel (engl, virus-like particles, VLPs), welche die genetische Information zur Expression des gewünschten Genprodukts enthalten und bei Einschleusung in mit Retrovirus-Partikeln (Virionen) transduzierbare Zellen zur Expression des vom Transfervektor codierten Such host cells (also referred to as packaging cell lines) thus 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
Gen produkts führen. Durch das jeweilige Retrovirus transduzierbare Zellen sind einem Fachmann bekannt. So sind z.B. aufgrund der großen Wirtsbreite von Foamy Viren eine Vielzahl an Zellen zur Infektion mit Foamy-Virus-Partikeln geeignet und schließen Lead gene products. Cells which can be transduced by the respective retrovirus are known to a person skilled in the art. Thus, e.g. due to the large host range of Foamy viruses a variety of cells suitable for Infection with foamy virus particles and close
beispielsweise Säugetier-, Vogel-, Reptilien- und Amphibien-Zellen ein. Bevorzugte Zellen zur Infektion mit retroviralen Partikeln wie bspw. Foamy-Virus-Partikeln sind humane Zellen wie beispielsweise die in den unten aufgeführten Beispielen verwendete Zelllinie HT1080. 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.
Die erfindungsgemäßen Gegenstände werden vorteilhafter Weise zu Kits The articles according to the invention advantageously become kits
zusammengestellt, mit denen die Expression eines gewünschten Genprodukts wie
bspw. eines heterologen, insbesondere nicht-retroviralen wie bspw. eines Nicht- Foamy-Virus-Proteins bewerkstelligt werden kann. So umfasst unter einem weiteren Gesichtspunkt der Erfindung ein Kit zur Expression eines erfindungsgemäßen Genprodukts wie bspw. eines in Bezug auf das betreffende Retrovirus heterologen, vorzugsweise nicht-retroviralen Proteins wie eines Nicht-Foamy-Virus-Proteinscompiled with which the expression of a desired gene product such as For example, a heterologous, especially non-retroviral such as a non-foamy virus protein can be accomplished. Thus, in a further aspect of the invention, 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
(i) ein wie vorstehend definiertes Vektorsystem und (i) a vector system as defined above, and
(ii) Zellen, die als wie vorstehend definierte Verpackungszellen geeignet sind. (ii) Cells suitable as packaging cells as defined above.
In einer weiteren Ausführungsform umfasst das erfindungsgemäße Kit außerdem (iii) Zellen, die zur Infektion mit Retrovirus-Partikeln wie z.B. Foamy-Virus- Partikeln geeignet sind. In diesem Zusammenhang ist noch einmal darauf hinzuweisen, dass derartige Zellen vorzugsweise auch durch entsprechende VLPs des betreffenden Virus transduzierbar sind. Das erfindungsgemäße Kit kann somit auch wie vorstehend definierte Wirtszellen (Verpackungszellen) zusammen mit den zur Infektion mit Retrovirus-Partikeln geeigneten Zellen enthalten. In a further embodiment, 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. In this connection, it should again be pointed out that such cells are preferably also transduceable by corresponding VLPs of the virus in question. 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.
Durch die Verwendung des erfindungsgemäßen Vektorsystems in Verbindung mit geeigneten Zellen ergibt sich als weiterer Gegenstand der vorliegenden Erfindung auch ein Verfahren zur Expression des jeweils gewünschten Genprodukts wie z.B. eines heterologen, insbesondere nicht-retroviralen wie bspw. Nicht-Foamy-Virus- Proteins in einer Zelle, das die Schritte By using the vector system according to the invention in conjunction with suitable cells, 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
(A) Transfizieren einer Zelle mit einem Vektorsystem gemäß der vorliegenden Erfindung, bei dem der Transfervektor eine in die Expressionskassette funktional eingefügte Nukleotidsequenz enthält, die für mindestens ein Genprodukt , insbesondere ein in Bezug auf das Retrovirus heterologes Genprodukt, wie bspw. ein nicht-retrovirales Protein codiert, wobei die Nukleotidsequenz auch für ein Reporterprotein oder -peptid codieren kann, und wobei die Zelle zur Bildung und Ausschleusung von Retrovirus-Partikeln (A) transfecting a cell with a vector system according to the present invention, in which the transfer vector contains a nucleotide sequence functionally inserted into the expression cassette which represents at least one gene product, in particular a gene product heterologous with respect to the retrovirus, such as a non-retroviral Protein, wherein the nucleotide sequence can also code for a reporter protein or peptide, and wherein the cell for the formation and discharge of retrovirus particles
(z.B. Foamy-Virus-Partikeln) aufgrund der Expression mindestens der essentiellen Virus-Proteine über den/die Vektor(en) gemäß Bestandteil (a) bzw. (1 ) der erfindungsgemäßen Vektorsysteme (also z.B. Gag- und Pol(e.g., foamy virus particles) 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
io
Proteine eines Foamy Virus über die Vektoren gemäß (a1 ) beziehungsweise den Vektor gemäß (a2) nach obiger Definition) ausgelegt ist, io Proteins of a foamy virus via the vectors according to (a1) or the vector according to (a2) as defined above) is designed,
Kultivieren der transfizierten Zelle in einem Kulturmedium unter Bedingungen, die die Bildung und Ausschleusung von Retrovirus-Partikeln in das Culturing the transfected cell in a culture medium under conditions that prevent the formation and discharge of retrovirus particles into the culture medium
Kulturmedium erlauben, Allow culture medium,
Gewinnen eines Retrovirus-Partikel enthaltenen Überstands aus der Kultur von Schritt (B) und Recovering a supernatant containing retrovirus particles from the culture of step (B) and
Transduzieren von Zellen, die zur Infektion mit Retrovirus-Partikeln geeignet sind, mit dem gegebenenfalls verdünnten Kulturüberstand von Schritt (C), umfasst. Transducing cells suitable for infection with retrovirus particles with optionally diluted culture supernatant from step (C).
Geeignete Bedingungen und Kulturmedien gemäß Schritt (B) sind einem Fachmann bekannt und können beispielsweise der neuestens Ausgabe von Ausubel et al. (Ed.) „Current Protocols in Molecular Biology", Wiley, New York, entnommen werden. Diesbezüglich wird für Foamy-Virus-Systeme der vorliegenden Erfindung 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
insbesondere auch auf die Beispiele in WO-A-2010/1 1 1608 verwiesen. Gleiches gilt entsprechend auch für die zur Infektion mit Foamy-Virus-Partikeln geeigneten Zellen. in particular also referred to the examples in WO-A-2010/1 1 1608. The same applies accordingly to the cells suitable for infection with foamy virus particles.
Ein weiterer Gegenstand der vorliegenden Erfindung betrifft ein Verfahren zur Expression eines wie vorliegend definierten Genprodukts, vorzugsweise eines nicht- retroviralen Genprodukts wie einem nicht-retroviralen, insbesondere Nicht-Foamy- Virus-Protein in einem Wirtsorganismus, umfassend das Einbringen einer wie vorstehend definierten Wirtszelle (Verpackungszelle), die zur Bildung und 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
Ausschleusung von Retrovirus-Partikeln (z.B. Foamy-Virus-Partikeln) aufgrund der Expression mindestens der essentiellen Virus-Proteine über den/die Vektor(en) gemäß Bestandteil (a) bzw. (1 ) der erfindungsgemäßen Vektorsysteme (also z.B. Gag- und Pol-Proteine eines Foamy Virus über die Vektoren gemäß (a1 ) Removal of retrovirus particles (eg foamy virus particles) due to the expression of 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)
beziehungsweise den Vektor gemäß (a2) nach obiger Definition) ausgelegt ist, in den Wirtsorganismus, wobei ein oder mehrere Zelle(n) des Wirtsorganismus durch Retrovirus-Partikel wie z.B. Foamy-Virus-Partikel transduzierbar ist/sind. or the vector according to (a2) as defined above) into the host organism, wherein one or more cells of the host organism are immobilized by retrovirus particles such as e.g. Foamy virus particles is / are transduable.
Der Wirtsorganismus kann ein humaner oder nicht-humaner, insbesondere The host organism may be a human or non-human, in particular
Säugetier-Wirtsorganismus sein. Die Expression des Genprodukts wie eines heterologen Genprodukts, insbesondere eines nicht-retroviralen, vorzugsweise Nicht-
Foamy-Virus-Proteins in dem Wirtsorganismus kann vorteilhafter Weise dazu genutzt werden, einen genetischen Defekt des Wirtsorganismus zu behandeln. Bspw. ist es so möglich, ein in dem Wirtsorganismus aufgrund eines genetischen Defekts nicht oder unzureichend exprimiertes Protein durch Expression über das Vektorsystem der vorliegenden Erfindung dem Wirtsorganismus in ausreichender Menge Being a mammalian host. Expression of 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. For example. Thus, it is possible for 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
bereitzustellen. Gleiches gilt auch für eine aufgrund eines genetischen Defekts exprimierte Proteinmutante, das die natürliche Funktion des Wildtyp-Proteins nicht oder nur in unzureichender Weise ausübt. In weiteren bevorzugten provide. The same applies to a protein mutant expressed as a result of a genetic defect, which does not or only inadequately exerts the natural function of the wild-type protein. In further preferred
Ausführungsformen der Erfindung kann durch Expression von RNAi auslösenden Genprodukten (z.B. siRNA, shRNA, miRNA, pre-miRNA, pri-miRNA) in dem 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
Wirtsorganismus eine Genregulation durchgeführt werden, um bspw. ein mit einer Erkrankung assoziiertes Gen herunterzuregulieren bzw. abzuschalten. A gene regulation can be carried out in order to downregulate, for example, a gene associated with a disease.
Die Figuren zeigen: The figures show:
Fig. 1 : zeigt eine schematische Darstellung verschiedener retroviraler Fig. 1: shows a schematic representation of various retroviral
Replikationsstrategien. Replication strategies.
Fig. 2 zeigt eine schematische Darstellung unterschiedlicher retroviraler Fig. 2 shows a schematic representation of different retroviral
Vektorsysteme zur transienten Transgenexpression. Vector systems for transient transgene expression.
Fig. 3 zeigt graphische Darstellungen der durchflusszytometrischen Analyse des zeitlichen Verlaufs der Transgenexpression verschiedener FV3 shows graphical representations of the flow cytometric analysis of the time course of transgene expression of various FVs
Vektoren in Zielzellen. Vectors in target cells.
PFV Vektorüberstände wurden durch transiente Transfektion von 293T PFV vector supernatants were transiently transfected by 293T
Zellen unter Verwendung eines expressions-optimierten 4- Komponenten PFV Vektorsystems hergestellt. Dieses Vektorsystem besteht aus dem Transfervektor puc2MD9, der eine interne SFFV U3 getriebene EGFP Expressionskassette enthält, sowie den Verpackungskonstrukten pcoPE (Env), pcoPG4 (Gag) and pcoPP (Pol). Die Vektorpartikel unterscheiden sich in der Art des kotransfizierten Pol Verpackungskonstruktes. Wt: pcoPP (wt, alle enzymatischen Aktivitäten intakt); pcoPPI (iPR, enzymatisch inaktivierte Protease); pcoPP2 (iRT, enzymatisch inaktivierte Reverse Transkriptase); pcoPP3 (ilN,
enzymatisch inaktivierte Integrase). Anschließend wurden HT1080 Zielzellen mit unverdünntem viralen Vektorüberstand transduziert und die EGFP Expression in den Zellpopulationen mittels Cells 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). Subsequently, HT1080 target cells were transduced with undiluted viral vector supernatant and EGFP expression in the cell populations by means of
Durchflusszytometrie im zeitlichen Verlauf analysiert. Flow cytometry analyzed over time.
Virale Partikel mit enzymatisch inaktivierter Protease (iPR) oder Reversen Transkriptase (iRT) ermöglichen eine schwache und kurz anhaltenden transienten EGFP Expression. Partikel mit einer enzymatisch inaktivierten Integrase (ilN) führen zu einer stärkeren aber weitestgehend transienten EGFP Expression wohingegen wildtypische Partikel zu einer starken konstitutiven Transgenexpression führen. zeigt die Eigenschaften verschiedener Ausführungsformen von PFV- Transfervektorkonstrukten der vorliegenden Erfindung. 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.
(A) Schematische Darstellung der Transfervektoren pMD9, SFFV U3 EGFP PFV3' und des erfindungsgemäßen Vektors SFFV U3 EGFP. pMD9: Standard PFV-Transfervektor mit einer internen SFFV-U3- getriebenen EGFP-Transgenkassette und essentiellen PFV eis- aktiven Verpackungssequenzen (CAS I, CAS II); SFFV U3 EGFP PFV 3': SFFV U3-getriebene EGFP-Expressionsvektoren der 3'-Teile des PFV- Vektorgenoms enthält, jedoch keine c/s-aktiven Verpackungssequenzen aufweist; SFFV U3 EGFP: SFFV U3-getriebener EGFP- Expressionsvektor, der keinerlei PFV-Sequenzen enthält. (A) 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.
(B) FACS Dotblot Profile von HT1080 Zielzellen 48h nach Transduktion mit unverdünntem zellfreiem 293T Überstand. MFI: Mittlere (B) FACS dot blot profiles of HT1080 target cells 48h after transduction with undiluted cell-free 293T supernatant. MFI: Medium
Fluoreszenzintensität. Fluorescence intensity.
(C) Es wurden HT1080 Zielzellen mit unverdünntem viralen (C) There were HT1080 target cells with undiluted viral
Vektorüberstand transduziert, und die stabile als auch transiente EGFP- Transgenexpression durch Durchflusszytometrie bestimmt. Die Vector supernatant transduced, and the stable as well as transient EGFP transgene expression determined by flow cytometry. The
Partikelfreisetzung und Pol-Verpackung wurde durch Western blot von Partikel- und Zelllysaten überprüft. Die Viruspartikel-assoziierte Particulate release and pol packaging was checked by Western blotting of particle and cell lysates. The virus particle associated
Nukleinsäurezusammensetzung wurde mittels qPCR bestimmt. Gezeigt sind die jeweiligen Werte, ausgehend von den jeweiligen Nucleic acid composition was determined by qPCR. Shown are the respective values, starting from the respective values
Transfervektoren.
zeigt den Einfluss von transgenen Transkriptionskontrollelementen auf die RNA-Transfereffizient. Transfer vectors. shows the influence of transgenic transcriptional control elements on RNA transfer efficiency.
(A) Schematische Darstellung der Transfervektoren pSFFV U3 EGFP und pcziEGqP (CMV immediate early Promoter/Enhancer Intron A). (A) Schematic representation of the transfer vectors pSFFV U3 EGFP and pcziEGqP (CMV immediate early promoter / enhancer intron A).
(B) PFV-Vektoren wurden durch transiente Transfektion von 293T- Zellen unter Verwendung eines Codon-optimierten 4-Komponenten PFV- Vektorsystems gemäß WO-A-2010/1 1 1608 unter der Verwendung der Transfervektoren gemäß (A) hergestellt. Nachfolgend wurden HT1080-Zielzellen mit unverdünntem Virusvektorüberstand oder einer(B) 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
1 :10-Verdünnung davon transduziert und die EGFP- Transgenexpression durch Durchflusszytometrie zeitabhängig gemessen. Transduced 1: 10 dilution thereof and measured EGFP transgene expression by time-dependent flow cytometry.
(C) Durchschnittliche EGFP-Fluoreszenzintensität der gesamten Zielzellenpopulation über die Zeit. (C) Average EGFP fluorescence intensity of the entire target cell population over time.
Zeitverlauf der transienten EGFP-Expression in transduzierten HT1080- Zellen. PFV-Vektoren wurden durch transiente Transfektion von 293T- Zellen unter Verwendung eines expressionsoptimierten 4-Komponenten PFV- Vektorsystems gemäß WO-A-2010/1 1 1608 unter Verwendung des pcziEGgP-Transfervektors der vorliegenden Erfindung wie bei Fig. 3 beschrieben hergestellt. Nachfolgend wurden HT1080-Zielzellen mit unverdünntem Virusvektorüberstand transduziert und die EGFP- Transgenexpression zeitabhängig durch Durchflusszytometrie gemessen. Time course of transient EGFP expression in transduced HT1080 cells. 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.
(A) Übereinandergelegte Histogramme der EGFP-Expression über die Zeit. (A) Overlapped histograms of EGFP expression over time.
(B) Durchschnittliche EGFP-Fluoreszenzintensität der gesamten Zielzellpopulation über die Zeit. Fig. 7: Vergleich der transienten und stabilen Gentransfereffizienz durch (B) Average EGFP fluorescence intensity of the entire target cell population over time. Fig. 7: Comparison of the transient and stable gene transfer efficiency by
retrovirale Vektorsysteme, die von verschiedenen Retrovirusspezies abgeleitet sind. Retrovirale Vektoren wurden durch transiente Transfektion von 293T-Zellen unter Verwendung eines Codon- optimierten 4-Komponenten PFV-Vektorsystems (PFV) oder
konventionellen Maus Leukämie Virus (MLV) oder Humanen 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
Immundefizienz Virus (HlV)-basierten Vektorsystemen unter Immunodeficiency virus (HIV) -based vector systems under
Verwendung von Transfervektoren der vorliegenden Erfindung zur transienten (A) oder stabilen (B) Transgenexpression erzeugt. Use of transfer vectors of the present invention for transient (A) or stable (B) transgene expression generated.
Nachfolgend wurden HT1080-Zielzellen mit unverdünntem viralen Vektorüberstand im Falle der transienten Transfervektoren (A) oder einer Serie 10-facher Verdünnungen für die stabilen Transfervektoren (B) transduziert und die EGFP-Transgenexpression zeitabhängig durch Durchflusszytometrie gemessen. Die Titer der stabilen Subsequently, 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. The titers of stable
Transfervektorüberstände wurden aus den Tag 3 Postinfektion d3- Durchflusszytometriedaten berechnet. Transfer vector supernatants were calculated from day 3 post infection d3 flow cytometry data.
Bestimmung essentieller viraler Komponenten für den stabilen bzw. transienten Gentransfer. Retrovirale Vektoren wurden durch transiente Transfektion von 293T-Zellen unter Verwendung von verschiedenen Kombinationen aus Verpackungsplasmiden und Transfervektoren für eine stabile (MD9 qP) oder transient Markergenexpression (EGqP) hergestellt. Es wurden Verpackungskonstrukte für wildtypisches Gag (Gag wt), Pol (Pol wt) und Env (Env wt) wie auch Pol Varianten mit enzymatisch inaktivierter RT Domäne (Pol iRT) oder Integrase Domäne (ilN) und Env Varianten für fusions-inaktives Hüllprotein (Env iSU/TM) in einzelnen Kombinationen verwendet. Nachfolgend wurden HT1080- Zielzellen mit seriell verdünntem Vektorüberstand transduziert und die EGFP-Transgenexpression zeitabhängig durch Durchflusszytometrie gemessen. A) Die Titer der Transfervektorüberstände wurden aus den Tag 2 Postinfektion-Durchflusszytometriedaten berechnet. Dargestellt sind die relativen Infektiositäten im Vergleich zum authentischen 4- Komponenten PFV Vektorsystem. B) Mittlere Fluoreszenzintensität der gesamten Zielzellpopulation 48 h nach Transduktion mit unverdünntem Vektorüberstand. Wt: wildtypisches Plasmid; -: entsprechendes Determination of essential viral components for stable or transient gene transfer. 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. Subsequently, HT1080 target cells were serially transduced with serially diluted vector supernatant and the EGFP transgene expression was measured time-dependently by flow cytometry. A) Titer of transfer vector supernatants were calculated from day 2 post infection flow cytometry data. Shown are the relative infectivities compared to the authentic 4-component PFV vector system. B) Mean fluorescence intensity of the entire target cell population 48 h after transduction with undiluted vector supernatant. Wt: wild-type plasmid; -: corresponding
Verpackungsplasmid wurde durch pUC19 DNA ersetzt. Packaging plasmid was replaced with pUC19 DNA.
RNA Transfer-vermittelte Cre Rekombinase Expression in R26R-YFP Maus Embryo Fibroblasten. PFV Vektorüberstände wurden durch
transiente Transfektion von 293T-Zellen unter Verwendung eines expressions-optimierten 4-Komponenten PFV-Vektorsystems gemäß WO-A-2010/1 1 1608 unter Verwendung von verschiedenen retroviralen (MD9 qP, MD9 Cre) oder RNA (EGqP, Cre) Transfervektoren der vorliegenden Erfindung wie unter Fig. 3 beschrieben hergestellt. NachRNA 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. To
Transduktion von R26R-YFP embryonalen Maus Fibroblasten (MEF) mit unverdünnten zellfreien Vektorüberständen wurde die EGFP/EYFP Expression im zeitlichen Verlauf durchflusszytometrisch analysiert. Dargestellt ist A) der Prozentsatz markergenpositiver Zellen und B) die mittlere Fluoreszenzintensität (MFI) der ganzen Zellpopulation. Transduction of R26R-YFP embryonic mouse fibroblasts (MEF) with undiluted cell-free vector supernatants EGFP / EYFP expression was analyzed by flow cytometry over time. Shown is A) the percentage of marker-positive cells and B) the mean fluorescence intensity (MFI) of the whole cell population.
Fig.10 Verlauf der transienten EGFP-Expression im Zeitraum von 1 bis 72 FIG. 10 Course of the transient EGFP expression in the period from 1 to 72. FIG
Stunden nach Transduktion. PFV-Vektoren wurden durch transiente Transfektion von 293T-Zellen unter Verwendung eines Hours after transduction. PFV vectors were generated by transient transfection of 293T cells using a
expressionsoptimierten 4-Komponenten PFV-Vektorsystems gemäß Expression-optimized 4-component PFV vector system according to
WO-A-2010/1 1 1608 unter Verwendung der Transfervektoren puc2MD9 EGqP (MD9), pSFFVU3 EGqP (SFFV EGqP) oder pcziEGqP (CMV EGqP) der vorliegenden Erfindung wie bei Fig. 3 beschrieben hergestellt. Die Vektorpartikel unterscheiden sich auch in der Art des kotransfizierten Pol-Verpackungskonstruktes. Wt: pcoPP (wt, alle enzymatischen Aktivitäten intakt); pcoPP2 (iRT, enzymatisch inaktivierte Reverse Transkriptase); pcoPP3 (ilN, enzymatisch inaktivierte Integrase). Nachfolgend wurden HT1080-Zielzellen mit unverdünntem Virusvektorüberstand durch Spinokulation (30 min, 10°C, 1200g) zu verschiedenen Zeitpunkten nach Aussaat transduziert und die EGFP-Transgenexpression der verschiedenen Proben, die zum gleichen Zeitpunkt geerntet wurden, mittels Durchflusszytometrie gemessen. (A) Schematische Übersicht des zeitlichen Ablaufs des Experimentes. (B) Mittlere EGFP-Fluoreszenzintensität (EGFP MFI) der verschiedenen Zielzellproben nach Transduktion mit unverdünntem zellfreiem 293T-Überstand in Abhängigkeit vom Zeitpunkt nach WO-A-2010/1 1 1608 using the transfer vectors puc2MD9 EGqP (MD9), pSFFVU3 EGqP (SFFV EGqP) or pcziEGqP (CMV EGqP) of the present invention as described in FIG. 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). Subsequently, 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
Beendigung der Inkubation mit Virusüberstand.
Verlauf der transienten EGFP-Expression im Zeitraum von 2 bis 16 Tagen nach Transduktion. PFV-Vektoren wurden durch transiente Transfektion von 293T-Zellen unter Verwendung eines Termination of incubation with virus supernatant. Course of transient EGFP expression in the period from 2 to 16 days after transduction. PFV vectors were generated by transient transfection of 293T cells using a
expressionsoptimierten 4-Komponenten PFV-Vektorsystems gemäß WO-A-2010/1 1 1608 unter Verwendung der Transfervektoren puc2MD9 EGqP (MD9), pSFFVU3 EGqP (SFFV EGqP) oder pcziEGqP (CMV EGqP) der vorliegenden Erfindung wie bei Fig. 3 beschrieben hergestellt. Die Vektorpartikel unterscheiden sich auch in der Art des kotransfizierten Pol-Verpackungskonstruktes. Wt: pcoPP (wt, alle enzymatischen Aktivitäten intakt); pcoPP2 (iRT, enzymatisch inaktivierte Reverse Transkriptase); pcoPP3 (ilN, enzymatisch inaktivierte Integrase). Nachfolgend wurden HT1080-Zielzellen mit unverdünntem Virusvektorüberstand durch Spinokulation (30 min, 10°C, 1200g) transduziert und die EGFP-Transgenexpression zeitabhängig mittels Durchflusszytometrie gemessen. (A) Schematische Übersicht des zeitlichen Ablaufs des Experimentes. (B) Mittlere EGFP- Fluoreszenzintensität (EGFP MFI) der verschiedenen Zielzell-Proben nach Transduktion mit unverdünntem zellfreiem 293T-Überstand in Abhängigkeit vom Zeitpunkt nach Beendigung der Inkubation mit Virusüberstand. expression-optimized 4-component PFV vector system according to WO-A-2010/11608 using the transfer vectors puc2MD9 EGqP (MD9), pSFFVU3 EGqP (SFFV EGqP) or pcziEGqP (CMV EGqP) of the present invention as described in FIG. 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). Subsequently, 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 Verlauf der Intensität des EGFP-Fluoreszenzsignals im Zeitraum von 1 bis 24 Stunden nach Transduktion mit verschiedenen EGFP- exprimierenden bzw. EGFP-markierten PFV Vektorpartikeln. PFV- Vektoren wurden durch transiente Transfektion von 293T-Zellen unterFIG. 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
Verwendung eines expressionsoptimierten 4-Komponenten PFV- Vektorsystems gemäß WO-A-2010/1 1 1608 unter Verwendung der Transfervektoren puc2MD9 EGqP (MD EG) oder pcziEGqP (CMV EG) sowie des leeren Vektors pczi (CMV) der vorliegenden Erfindung wie bei Fig. 3 beschrieben hergestellt. Die Vektorpartikel unterscheiden sich auch bezüglich des kotransfizierten Gag Verpackungskonstruktes: Use of an expression-optimized 4-component PFV vector system according to WO-A-2010/11608 using the transfer vectors puc2MD9 EGqP (MD EG) or pcziEGqP (CMV EG) and the empty vector pczi (CMV) of the present invention as in FIG. 3 described manufactured. The vector particles also differ with respect to the co-transfected gag packaging construct:
pcoPG4 (p71 , authentisches Volllängen-PFV p71 Gag); pcoPG4 pcoPG4 (p71, authentic full-length PFV p71 gag); pcoPG4
CEGFP (p71 EG, Volllängen-PFV p71 Gag mit C-terminaler EGFP- Fusion); pcoPG4 1 -621 CEGFP (p68 EG, C-terminal verkürztes PFV
p68Gag mit C-terminaler EGFP-Fusion); Abwesenheit von Gag 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
Verpackungskonstrukt (- /). Bei einigen Proben wurde nur der Packaging construct (- /). For some samples only the
Transfervektor pcziEGqP (- / CMV EG) transfiziert. Nachfolgend wurden HT1080-Zielzellen mit unverdünntem Virusvektorüberstand durch Spinokulation (30 min, 10°C, 1200g) zu verschiedenen Zeitpunkten nach Aussaat in Anwesenheit (gestrichelte Linien) oder Abwesenheit (durchgezogene Linien) von 100 g/ml Cycloheximid transduziert und die EGFP-Transgenexpression der verschiedenen Proben, die zum gleichen Zeitpunkt geerntet wurden, mittels Durchflusszytometrie gemessen. Als Kontrolle wurden HT1080 Zielzellen verwendet, die nicht mit zellfreiem 293T-Überstand inkubiert wurden (- / - (mock)). (A) Cycloheximidstruktur und Information zur Wirkungsweise. (B) Transfer vector pcziEGqP (- / CMV EG) transfected. Subsequently, 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)
Aufstellung der verwendeten Kombinationen von Gag Verpackungsund Transfervektor Komponenten. (C) Schematische Übersicht des zeitlichen Ablaufs des Experimentes. (D) RNA-Transfer. Mittlere EGFP- Fluoreszenzintensität (EGFP MFI) der verschiedenen Zielzellproben nicht-EGFP-markierter PFV-Partikel nach Transduktion mit List of used combinations of Gag packaging and transfer vector components. (C) Schematic overview of the time course of the experiment. (D) RNA transfer. Mean EGFP fluorescence intensity (EGFP MFI) of the different target cell samples of non-EGFP-labeled PFV particles after transduction with
unverdünntem zellfreiem 293T-Überstand in Abhängigkeit vom undiluted cell-free 293T supernatant depending on
Zeitpunkt nach Beendigung der Inkubation mit Virusüberstand. (E) Protein-Transfer. Mittlere EGFP-Fluoreszenzintensität (EGFP MFI) der verschiedenen Zielzellproben EGFP-markierter PFV-Partikel nach Transduktion mit unverdünntem zellfreiem 293T-Überstand in Time after incubation with virus supernatant. (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
Abhängigkeit vom Zeitpunkt nach Beendigung der Inkubation mit Virusüberstand. Dependence on the time after completion of the incubation with virus supernatant.
Dosisabhängigkeit des RNA-Transfers. PFV-Vektoren wurden durch transiente Transfektion von 293T-Zellen unter Verwendung eines expressionsoptimierten 4-Komponenten PFV-Vektorsystems gemäß WO-A-2010/1 1 1608 unter Verwendung unterschiedlicher Mengen der Transfervektoren pcziEGgP (CMV) und pSFFVU3 EGqP (SFFV) der vorliegenden Erfindung wie bei Fig. 3 beschrieben hergestellt. Für alle Proben wurden die gleichen Mengen an Verpackungsplasmiden verwendet und die Gesamtmenge an eingesetzter DNA durch Dose-dependent RNA transfer. 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
Auffüllung mit pUC19-DNA konstant gehalten. 48 Stunden nach
Transfektion wurden die zellfreien viralen Uberstände geerntet. Filling with pUC19 DNA kept constant. 48 hours after Transfection, the cell-free viral supernatants were harvested.
Anschließend wurden aus den transfizierten 293T-Zellen die Gesamt- RNA extrahiert und die Kopienzahl an EGFP-mRNA in den einzelnen Proben mit einer EGFP-spezifischen quantitativen PCR bestimmt (Zell- RNA, gestrichelte Linien). Die Zell-RNA-Mengen wurden auf die Menge an isolierter Gesamt-RNA normalisiert. Außerdem wurden aus 10 ml Überstand virale Partikel durch Ultrazentrifugation pelletiert und anschließend die Proteinzusammensetzung mittels Western-Blot und die EGFP-mRNA-Verpackung (Virus-RNA, durchgezogene Linien) mit einer EGFP-spezifischen quantitativen PCR analysiert. Die Virus-RNA- Mengen wurden auf die Menge an detektierbarem Kapsid (Gag)-Protein normalisiert. Weiterhin wurden mit 1 ml Proben der verschiedenen unverdünnten Vektorüberstände HT1080-Zielzellen transduziert und die EGFP-Transgenexpression (MFI, gepunktete Linien) 48 Stunden später durch Durchflusszytometrie gemessen. Subsequently, the total 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. In addition, 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. Furthermore, 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.
Die vorliegende Erfindung wird durch die folgenden nicht-einschränkenden Beispiele näher erläutert: The present invention is further illustrated by the following non-limiting examples:
BEISPIELE Beispiel 1 Um die Notwendigkeit der foamyviralen enzymatischen Funktionen für die stabile genetische Modifikation von Zielzellen mittels foamyviraler Vektoren zu bestimmen, wurden mit einem Standard-PFV-Transfervektor (pMD9, Fig. 4A) verschiedene EGFP-Transgen enthaltende FV-Vektorpartikel produziert. Diese unterschieden sich in der Art des verwendeten FV-Pol-Verpackungsplasmids. Es wurden Partikel hergestellt, die ein wildtypisches Pol-Protein (wt), oder Pol-Protein-Varianten enthielten, die entweder eine enzymatisch inaktive Domäne der Protease (iPR), der Reversen Transkriptase (iR) oder der Integrase (ilN) beinhalteten. EXAMPLES 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).
Nach Transduktion von permissiven Zielzellen wurde die EGFP-Transgenexpression durchflusszytometrisch über einen Zeitraum von 14 Tagen analysiert (Fig. 3). Bei
Vektorüberständen, die Pol-Proteine mit inaktiver Integrase enthielten, wurde initial eine nahezu wildtypische Transgenexpression beobachtet, die aber innerhalb einer Woche auf eine stark verringerte stabile Expression zurückging. Diese Viren können ihr verpacktes RNA-Genom revers transribieren, allerdings nicht über die virale Enzymfunktion sondern nur über nicht-homologe Rekombinationsmechanismen (ähnlich stabil transfizierten Plasmiden) in das Wirtsgenom integrieren. Bei After transducing permissive target cells, EGFP transgene expression was analyzed by flow cytometry over a period of 14 days (Figure 3). at Vector supernatants containing Pol proteins with inactive integrase were initially observed to be almost wild-type transgene expression, but this decreased to a greatly reduced stable expression within one week. These viruses can reverse transribed their packaged RNA genome, but not via the viral enzyme function but only non-homologous recombination mechanisms (similar stable transfected plasmids) integrate into the host genome. at
Vektorüberständen, die Pol-Proteine mit inaktiver Protease oder inaktiver Reverser Transkriptase enthielten, wurde im Vergleich zu wildtypischen FV-Vektoren eine sehr schwache und nur wenige Tage anhaltende Transgenexpression gemessen. Diese Partikel sind nicht in der Lage, das verpackte RNA-Genom in DNA umzuschreiben. Die Transgenexpression erfolgt hier also als direkte Translation der verpackten RNA. Welche Art von verpackter RNA für die Transgenexpression translatiert wird ist unklar. Die genomische Volllängen-RNA, welche die essentiellen cis-aktiven viralen Sequenzen CASI und CASII enthält, sollte auf Grund der enthaltenden extensiven 5'- untranslatierten (UT-) Regionen nicht zu einer Transgenexpression führen (Fig. 4A). Auch die im FV 5' LTR initiierende, einfach gespleisste mRNA verfügt noch über große 5'-UT-Bereiche, so dass auch sie keine Transgenexpression ermöglichen sollte (Fig. 4A). Lediglich die vom internen Promotor initierende, ungespleisste mRNA der Transgen kassette verfügt über gutes translationales Potential (Fig 4A). Dies legt nahe, dass FV-Vektorpartikel neben der viralen genomischen RNA auch signifikante Mengen an subgenomischen mRNAs verpacken, die hauptsächlich zur transienten Transgenexpression von Vektoren beitragen, die keine reverse Vector supernatants containing inactive protease or inactive reverse transcriptase Pol proteins were compared to wild-type FV vectors for very low transgene expression lasting only a few days. These particles are unable to rewrite the packaged RNA genome into DNA. Thus, transgene expression occurs as direct translation of the packaged RNA. Which type of packaged RNA is translated for transgene expression is unclear. The full length genomic RNA containing the essential cis-active viral sequences CASI and CASII should not result in transgene expression due to the extensive 5 'untranslated (UT) regions containing it (Figure 4A). 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
Transkription der verpackten RNAs durchlaufen können. Can undergo transcription of the packaged RNAs.
Deshalb sollte getestet werden, ob FV-Vektorpartikel, denen in der Verpackungszelle nur Transgen-haltige RNAs ohne die essentiellen viralen cis-aktiven Sequenzen (CASI und CASII) angeboten werden, diese verpacken und zu deren Translation in Zielzellen führen können. Hierzu wurde neben den FV-Verpackungsplasmiden für Gag, Pol und Env während der Vektorproduktion Expressionsvektoren kotransfiziert, die entweder nur die im Standard PFV-Vektor pMD9 enthaltende Therefore, it should be tested whether 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. In addition to the 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
Transgenexpression kassette mit einem zusätzlichen Poly-Adenylierungssignal enthielten (pSFFV U3 EGFP) oder anstelle dessen über die 3'-LTR-Sequenzen des pMD9-Vektors (pSSFV U3 EGFP PFV 3') verfügten (Fig. 4A). Die Analyse der verschiedenen Vektorpartikel und der durch sie transduzierten Zielzellen ergab, dass die transiente Transgenexpression auf einem ähnlichen oder sogar leicht erhöhtem
Niveau lag wie sie bei einem Standard-Transfervektor und RT inaktivierter Polymerase gemessen wurde (Fig. 4B, C). Auch der Gehalt an EGFP-Gen haltiger RNA in den viralen Partikeln war nahezu identisch (Fig. 4C). Dieser Befund legt nahe, dass PFV-Partikel mRNAs, die keine authentischen PFV Sequenzen enthalten effizient verpacken und nach Transduktion in Zielzellen zu deren Translation bringen können. 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). Analysis of the various vector particles and the target cells transduced by them revealed that transient transgene expression was at a similar or even slightly elevated level Level as measured on a standard transfer vector and RT inactivated polymerase (Figure 4B, C). Also, 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.
Beispiel 2 Es ist bekannt, dass Spleissen von mRNAs mit der Bindung von snRNAs und des Spleissosom-Komplexes diese stabilisieren und für den nuklearen Export markieren kann. Um den Einfluss der Transkriptionskassette auf den FV-Kapsid-vermittelten RNA Transfer zu analysieren, wurden gleiche Mengen verschiedener EGFP- transgenhaltiger Expressionsvektoren mit PFV Gag, Pol und Env 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
Verpackungsplasmiden zur Vektorpartikelproduktion kotransfiziert (Fig. 5A). Nach Transduktion von Zielzellen mit verschiedenen Verdünnungen vektorhaltiger Packaging plasmids are cotransfected for vector particle production (Figure 5A). After transduction of target cells with different dilutions vector containing
Überstände wurde die Transgenexpression im zeitlichen Verlauf Supernatants were transgene expression over time
durchflusszytometrisch analysiert. Die Ergebnisse zeigen, dass die Zielzell- Transgenexpression (gekennzeichnet durch die gemessene MFI) durch analyzed by flow cytometry. The results show that the target cell transgene expression (indicated by the measured MFI) by
Vektorpartikel, die mit einem CMV Promoter - Intron getriebenen Transfervektor produziert wurden, zu allen Zeitpunkten wesentlich höher war als diejenige von Vektorpartikeln, die mit einem intronlosen, SFFVU3 Promoter getriebenen Transfer erreicht wurde (Fig. 5B, C). Die transiente Expression nahm bei den höchsten eingesetzten Virusmengen im zeitlichen Verlauf der Messungen innerhalb von 6 Tagen kontinuierlich bis auf negative Kontrollwerte ab (Fig. 5B, C). Vector particles produced with a CMV promoter-intron driven transfer vector were significantly higher at all times than that of vector particles achieved with an intronless SFFVU3 promoter driven transfer (Figure 5B, C). Transient expression decreased continuously over the course of the measurements within 6 days, down to negative control values, at the highest amounts of virus used (FIG. 5B, C).
Eine genauere Analyse des zeitlichen Verlaufs der transienten EGFP- Transgenexpression, die durch Vektorpartikel, die mit Hilfe eines CMV Promoter - Intron getriebenen Transfervektors hergestellt wurden, ermöglicht wurde, ergab, dass eine maximale Fluoreszenzintensität nach 36-48 Std beobachtet werden konnte (Fig. 6). Allerdings konnte bereits 1 Std. nach Transduktion eine deutliche A more detailed analysis of the time course of transient EGFP transgene expression, made possible by vector particles prepared by means of a CMV promoter-intron driven transfer vector, revealed that maximum fluorescence intensity could be observed after 36-48 h (FIG ). However, already 1 h after transduction a clear
Transgenexpression gemessen werden. Transgene expression can be measured.
Beispiel 3
Als nächstes wurde analysiert, ob andere retrovirale Vektorsysteme ebenfalls zu einem RNA-Transfer fähig sind, der unabhängig von viralen Sequenzen auf dem Transfervektor ist, und deren Effizienz mit den FV-Vektorsystemen verglichen. Hierzu wurden HIV-1 -, MLV- und PFV- Vektorpartikel durch Kotransfektion eines CMV Promoter - Intron getriebenen Transfervektors mit den jeweiligen Example 3 Next, 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. For this purpose, HIV-1, MLV and PFV vector particles were cotransfected by a CMV promoter-intron-driven transfer vector with the respective
Verpackungsplasmiden kotransfiziert und die Transgenexpression in Zielzellen nach Transduktion mit unverdünnten Vektorüberständen durchflusszytometrisch bestimmt (Fig. 7). Zum Vergleich wurden parallel analoge Vektorpartikel mit den 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
entsprechenden Standard-Transfervektoren für eine konstitutive, stabile corresponding standard transfer vectors for a constitutive, stable
Transgenexpression hergestellt, und deren Titer mittels durchflusszytometrischer Analyse 72 Std. nach Transduktion auf der gleichen Zielzellart bestimmt. Die Transgene expression and their titer determined by flow cytometric analysis 72 hrs after transduction on the same target cell type. The
Ergebnisse zeigen, dass absolut gesehen der PFV-vermittelte RNA-Transfer mindestens 3-fach stärker als der HIV-1 vermittelte ist, während nur ein sehr schwacher MLV-vermittelter RNA-Transfer gemessen wurde (Fig. 7A). Zieht man die Titer der analogen Vektorüberstände für eine stabile Transgenexpression als Maß für die physikalische Partikelmenge mit in die Betrachtung ein, so ist die RNA- Transfereffizienz pro Vektorpartikel bei PFV Vektoren im Vergleich zu derjenigen von HIV-1 etwa 50-fach höher (Fig. 7B). 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).
Beispiel 4 Example 4
Für einen stabilen Gentransfer mit konstitutiver Transgenexpression mittels retroviraler Vektoren sind neben dem Transfervektor, der über bestimmte essentielle cis-aktive Sequenz verfügen muss, Verpackungsplasmide der Strukturproteine Gag und Env, wie auch der Enzymaktivitäten des Pol Proteins, Protease, Reverse For a stable gene transfer with constitutive transgene expression by means of retroviral vectors, in addition to the transfer vector, which must have certain essential cis-active sequence packaging plasmids of the structural proteins Gag and Env, as well as the enzyme activities of the Pol protein, protease, reverse
Transkriptase und Integrase unabdingbar (Fig. 8A). Um zu bestimmen, welche Komponenten des PFV- Vektorsystems für den RNA-Transfer, der zu einer transienten Transgenexpression in Zielzellen führt, notwendig sind, wurde ein CMV Promoter - Intron getriebener Transfervektor mit verschiedenen Kombinationen von Verpackungsplasmiden kotransfiziert. Das Potential der so erzeugten Transcriptase and integrase indispensable (FIG. 8A). In order to determine which components of the PFV vector system are necessary for RNA transfer resulting in transient transgene expression in target cells, a CMV promoter-intron driven transfer vector was cotransfected with various combinations of packaging plasmids. The potential of the so generated
Vektorüberstände, eine transiente Markergenexpression in Zielzellen zu erzeugen, wurde anschließend mittels durchflusszytometrischer Analyse bestimmt (Fig. 8B). Die Ergebnisse zeigen, dass das FV-Kapsidprotein Gag essentiell ist. Allerdings spielt es
keine Rolle, ob nur das p71 Gag-Vorläufer, das p68Gag-Prozessierungsprodukt oder ein Gemisch aus beiden verwendet wurde. Gag alleine reichte allerdings nicht für eine Markergenexpression in Zielzellen aus. Für einen effizienten RNA-Transfer mussten Partikel neben Gag auch ein fusionsfähiges FV-Env-Protein enthalten. Env alleine konnte zwar einen RNA-Transfer in Zielzellen ermöglichen, die Effizienz der Vector supernatants to generate transient marker gene expression in target cells were subsequently determined by flow cytometric analysis (Figure 8B). The results show that the FV capsid protein Gag is essential. However, it does regardless of whether only the p71 gag precursor, the p68 gag processing product or a mixture of both was used. However, Gag alone was not sufficient for marker gene expression in target cells. For efficient RNA transfer, particles in addition to Gag also had to contain a fusion-capable FV Env protein. Although Env alone was able to facilitate RNA transfer into target cells, the efficiency of the
Transgenexpression war jedoch mindestens 150-fach geringer als in der However, transgene expression was at least 150-fold lower than in the
Anwesenheit von Gag. FV-Pol, seine Reverse-Transkriptase- oder Integrase-Aktivität hingegen waren für eine RNA-Transduktion nicht essentiell. Beispiel 5 Presence of gag. In contrast, FV-Pol, its reverse transcriptase or integrase activity were not essential for RNA transduction. Example 5
Sämtliche bisherigen Daten zeigen, dass ein effizienter Transfer und eine Translation EGFP-ORF-haltiger RNAs mittels FV-Vektorpartikeln in Zielzellen möglich ist. Um eine breitere Fähigkeit dieses Systems zu demonstrieren, wurden FV- Transfervektoren für eine stabile bzw. transiente Expression einer humanisierten Form der Cre-Rekombinase hergestellt, entsprechende virale Vektorüberstände produziert und die enzymatische Funktion in Zielzellen bestimmt (Fig. 9A). Es wurden embryonale Maus-Fibroblasten als Zielzellen transduziert, die eine LoxP flankierte EYFP-Expressionskassette im ROSA26-Locus enthielten, die nur nach erfolgter Cre-vermittelter Rekombination transkriptioneil aktiv wird (Srinivas 2001 ) (Fig. 9). Die Daten zeigen, dass auch ein Cre-Transgen effizient in Zielzellen transient funktionell exprimiert werden kann, das eine YFP-Markergenexpression nach genomischer Rekombination aktiviert. Der direkte EGFP-Markergentransfer mittels Standard-PFV-Transfervektoren führte zu einem stabilen Markergentransfer, während die EGFP-Expression nach RNA-Transfer kontinuierlich im All data to date show that efficient transfer and translation of EGFP-ORF-containing RNAs by FV vector particles is possible in target cells. To demonstrate a broader capability of this system, 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). The data show that even a Cre-transgene can be efficiently transiently functionally expressed in target cells that activate YFP marker gene expression following genomic recombination. Direct EGFP marker gene transfer using standard PFV transfer vectors resulted in stable marker gene transfer, whereas EGFP expression after RNA transfer was continuous in the
Beobachtungszeitraum abnahm. Observation period decreased.
Beispiel 6 Um den Einfluss der Transkriptionskassette sowie der Enzymaktivitäten des Pol- Proteins auf den transienten Gentransfer und den zeitlichen Verlauf der 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
Transgenexpression zu untersuchen, wurden zunächst PFV-Vektorüberstände durch transiente Transfektion von 293T-Zellen unter Verwendung eines Initially, PFV vector supernatants were screened for transgene expression by transient transfection of 293T cells using a
expressionsoptimierten 4-Komponenten, wie in Beispiel 1 beschrieben, PFV-
Vektorsystems hergestellt. Dabei wurden die Transfervektoren puc2MD9 EGqP (MD9), pSFFVU3 EGqP (SFFV EGqP) und pcziEGqP (CMV EGqP) (Fig. 4A bzw. Fig. 5) eingesetzt. Bei dem Vektor pcziEGqP handelt es sich um einen CMV- Promotor-Intron-getriebenen Transfervektor, während der Vektor pSFFVU3 EGqP einen intronlosen SFFVU3-Promotor aufweist. Die den Standard-PFV-Transfervektor puc2MD9 enthaltenden Vektorpartikel unterschieden sich zusätzlich in der Art des kotransfizierten Pol-Verpackungskonstruktes: Bei dem Wildtyp-Konstrukt pcoPP sind alle enzymatischen Aktivitäten intakt, das Konstrukt pcoPP2 (iRT) weist eine inaktivierte Reverse Transkriptase auf, das Konstrukt pcoPP3 (ilN) eine inaktivierte Integrase. Die produzierten Vektorüberstände wurden zur Transduktion von HT1080- Zielzellen durch Spinokulation (30 min, 10°C, 1200 g) verwendet. Die Transduktion erfolgte zu verschiedenen Zeitpunkten nach der Aussaat, die EGFP- Transgenexpression wurde mittels Durchflusszytometrie gemessen (Fig. 10A). Das Ergebnis der Durchflusszytometrie ergab, dass eine Transduktion der Zielzellen mit Viruspartikeln, die durch Transfektion der 293T-Zellen mit dem Standard PFV-expression-optimized 4-component, as described in Example 1, PFV Vector system produced. In this case, 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: 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-
Transfervektor puc2MD9 produziert wurden, und bei denen eine Kotransfektion mit dem Wildtyp-Pol-Verpackungskonstrukts (MD9 / wt) erfolgte, einen stetigen Anstieg der EGFP-Expression in den Zielzellen während der ersten 70 Stunden hervorrief (Fig. 10B). Eine Transduktion der Zellen mit den anderen Vektorpartikeln führte zu einem Anstieg der Expression innerhalb der ersten 12 Stunden. Ab diesem Zeitpunkt war die Expression innerhalb der nächsten 58 Stunden im Wesentlichen stabil. Am geringsten war die Zielzell-Transgenexpression, die durch Vektorpartikel mit dem intronlosen, SFFVU3-Promotor-getriebenen Transfervektor (SFFV EGqP) ausgelöst wurde, sowie die Expression, die durch Vektorpartikel hervorgerufen wurde, die mit dem Standardtransfervektor bei Kotransfektion eines Pol-Verpackungskonstrukts mit inaktivierter Reverser Transkriptase (MD9 / iRT) produziert wurden (Fig. 10B). Eine Messung der Expression über einen längeren Beobachtungszeitraum (Fig. 1 1 A) ergab, dass lediglich eine Transduktion mit Vektorpartikeln, die mit dem Standard PFV-Transfervektor puc2MD9 bei Kotransfektion mit dem Wildtyp-Pol- Verpackungskonstrukts (MD9 / wt) produziert wurden, eine stabile Expression über den gesamten Beobachtungszeitraum von 16 Tagen verursachen kann (Fig. 1 1 B). Eine Zielzell-Transgenexpression nach Transduktion mit Vektorpartikeln mit dem CMV-Promotor-Intron-getriebenen Transfervektor (CMV EGqP) bzw. dem intronlosen SFFVU3 Promotor-getriebenen Transfervektor (SFFV EGqP) war ab einem Zeitpunkt
von 5 bis 7 Tagen nach Infektion nicht mehr messbar (Fig. 1 1 B). Ebenso führte eine Kotransfektion des Standardtransfervektors mit einem Pol-Verpackungskonstrukt mit inaktivierter Reverser Transkriptase (MD9 / iRT) zu Vektorpartikeln, die einen stetigen Rückgang der Expression innerhalb der ersten 7 Tage nach Infektion bis auf das Niveau der Negativkontrolle (mock) auslösen (Fig. 1 1 B). Die Daten zeigen, dass die Expression, die durch Vektorpartikel mit den erfindungsgemäßen CMV-Promotor- Intron getriebenen und intronlosen SFFVU3-Promotor getriebenen Transfervektoren ausgelöst wird, vollständig transient ist. Eine Kotransfektion des Transfer vector puc2MD9, and co-transfected with the wild-type Pol packaging construct (MD9 / wt), produced a steady increase in EGFP expression in the target cells during the first 70 hours (Figure 10B). Transduction of the cells with the other vector particles resulted in an increase in expression within the first 12 hours. From this time point, expression was essentially stable within the next 58 hours. The lowest was the target cell transgene expression elicited by vector particles with the intronless SFFVU3 promoter-driven transfer vector (SFFV EGqP), as well as the expression induced by vector particles inactivated with the standard transfer vector upon cotransfection of a Pol packaging construct Reverse transcriptase (MD9 / iRT) were produced (Figure 10B). 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). A target cell transgene expression after transduction with vector particles with the CMV promoter-intron-driven transfer vector (CMV EGqP) or the intronless SFFVU3 promoter-driven transfer vector (SFFV EGqP) was from one point in time from 5 to 7 days after infection no longer measurable (Fig. 1 1 B). Similarly, cotransfection of the standard transfer vector with a reverse transcriptase inactivated polypack construct (MD9 / iRT) resulted in vector particles that induce a steady decline in expression within the first 7 days postinfection to the level of the negative control (mock) (FIG 1 B). The data show that the expression triggered by vector particles with the CMV promoter-intron driven and intronless SFFVU3 promoter driven transfer vectors is completely transient. A co-transfection of the
Standardtransfervektors mit einem Pol-Verpackungskonstrukt mit inaktivierter Integrase (MD9 / ilN) hingegen führte Vektorpartikeln, die zunächst zu einem Standard transfer vector with a Pol packaging construct with inactivated integrase (MD9 / ilN), however, led vector particles, which initially to a
Rückgang der Expression im Zeitraum von 2 bis 7 Tage nach Infektion führen, die auf einem 500 bis 1000-fach niedrigeren Niveau im Vergleich zum Decrease in expression in the period of 2 to 7 days after infection lead to a 500 to 1000-fold lower level compared to
Standardtransfervektor mit wildtypischem Pol-Verpackungskonstrukt (MD9 / wt) aber deutlich über dem der Negativkontrolle (mock) lag (Fig. 1 1 B). Ab diesem Zeitpunkt war die EGFP-Expression aber bis zum Ende des Beobachtungszeitraums von 16 Tagen stabil. Standard transfer vector with wild-type Pol packaging construct (MD9 / wt) but significantly above that of the negative control (mock) was (Fig. 1 1 B). From this point on, however, EGFP expression was stable until the end of the 16-day observation period.
Beispiel 7: In einem weiteren Versuch wurde das EGFP-Fluoreszenzsignal in den Zielzellen nach Transduktion mit verschiedenen EGFP-exprimierenden bzw. EGFP-markierten PFV-Vektorpartikeln über einen Zeitraum von 1 bis 24 Stunden nach Infektion gemessen. Die Herstellung der PFV- Vektorüberstände erfolgte durch transiente Transfektion von 293T-Zellen unter Verwendung eines expressionsoptimierten 4- Komponenten PFV-Vektorsystems (siehe Beispiel 1 ). Dabei wurden die 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
Transfervektoren puc2MD9 EGqP (MD EG), pcziEGqP (CMV EG) sowie der leere Vektor pczi (CMV) verwendet. Die mit dem Transfervektor pcziEGqP (CMV EG) hergestellten Partikel unterschieden sich zusätzlich in der Art des kotransfizierten Gag Verpackungskonstruktes. Es wurden die Konstrukte pcoPG4 (p71 , 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,
authentisches Volllängen-PFV p71 Gag), pcoPG4 CEGFP (p71 EG, Volllängen-PFV p71 Gag mit C-terminaler EGFP-Fusion), pcoPG4 1 -621 CEGFP (p68 EG, C-terminal verkürztes PFV p68Gag mit C-terminaler EGFP-Fusion) oder aber kein Gag- Verpackungskonstrukt kotransfiziert. Die produzierten Vektorüberstände wurden zur Transduktion von HT1080-Zielzellen durch Spinokulation (30 min, 10°C, 1200 g)
verwendet. Die Transfektion erfolgte zu verschiedenen Zeitpunkten nach der 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 ) or 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
Aussaat, die EGFP-Transgenexpression wurde mittels Durchflusszytometrie gemessen (Fig. 12C). Die Messung der EGFP-Fluoreszenzintensität in Zielzellen, die mit EGFP-exprimierenden PFV-Vektorpartikeln transduziert wurden, ergab, dass die Zielzell-Transgenexpression nach Transduktion mit den Vektorpartikeln, die wildtypisches Gag und den Transfervektor puc2MD9 EGqP (MD EG) bzw. den Transfervektor pcziEGqP (CMV EG) enthalten, nach 24 Stunden nahezu gleich hoch war, wobei die CMV EG-Vektorpartikel anfänglich zu einer höheren Expression führten (Fig. 12D, durchgezogene Linien). In Kontrollansätzen, in denen das die Proteinbiosynthese in eukaryotischen Zellen hemmende Antibiotikum Cycloheximid (Fig. 12A) zugesetzt wurde, wurde eine deutlich geringere Fluoreszenz gemessen, die während des Beobachtungszeitraums nicht anstieg (Fig. 12D, gestrichelte Sowing, EGFP transgene expression was measured by flow cytometry (Figure 12C). Measurement of EGFP fluorescence intensity in target cells transduced with EGFP-expressing PFV vector particles revealed that target cell transgene expression after transduction with the vector particles, the wild-type Gag and the transfer vector puc2MD9 EGqP (MD EG) and the transfer vector pcziEGqP, respectively (CMV EG) was nearly equal after 24 hours, with the CMV EG vector particles initially leading to higher expression (Figure 12D, solid lines). In control batches in which the antibiotic cycloheximide inhibiting protein biosynthesis in eukaryotic cells (FIG. 12A) was added, a significantly lower fluorescence was measured, which did not increase during the observation period (FIG. 12D, dashed line)
Linien). Die gemessene Fluoreszenz ist somit auf die EGFP-Expression nach Lines). The measured fluorescence is thus indicative of EGFP expression
Transduktion mit den EGFP-exprimierenden Vektorpartikeln zurückzuführen. Attributed to transduction with the EGFP-expressing vector particles.
Der Protein-Transfer wurde durch Messung der EGFP-Fluoreszenz Intensität von Zielzellen, die mit EGFP-Kapsid-markierten PFV-Vektorpartikeln transduziert wurden, bestimmt (Fig. 12E, durchgezogene Linien). Die Messung ergab, dass Vektorpartikel, die bei einer Kotransfektion mit einem terminal verkürzten PFV p68Gag (p68 EG) entstehen, zu eine stärkeren Zielzell-Fluoreszenz als Ergebnis des Proteintransfers zeigen, als Vektorpartikel, die bei einer Kotransfektion mit einem Volllängen-PFV p71 Gag (p71 EG) produziert werden. Weiterhin konnte gezeigt werden, dass die Fluoreszenz, die durch eine Transduktion mit EGFP-markierten, kapsidlosen 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
Vektorpartikeln ausgelöst wird, bei denen kein Gag-Verpackungskonstrukt Vector particles are triggered in which no gag packaging construct
kotransfiziert wurde (CMV EG), deutlich geringer ist als die Fluoreszenz, die durch Transduktion mit EGFP-Kapsid-markierten Vektorpartikeln (p71 EG, p68 EG) ausgelöst wird. Da die Fluoreszenz in den mit EGFP-markierten PFV-Vektorpartikeln transduzierten Zellen von dem Transfer der EGFP-markierten Partikel, und nicht von dem Transfer und der anschließenden Expression der RNA in der Zielzelle abhängt, hatte eine Zugabe von Cycloheximid zu den Infektionsansätzen in diesem Fall keinen Einfluss auf die gemessene Fluoreszenz-Intensität (Fig. 12E, gestrichelte Linien). cotransfected (CMV EG), is significantly lower than the fluorescence, which is triggered by transduction with EGFP-capsid-labeled vector particles (p71 EG, p68 EG). Since 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).
Beispiel 8:
Um die Dosisabhängigkeit des RNA-Transfers nachzuweisen, wurden zunächst PFV- Vektoren durch transiente Transfektion von 293T-Zellen unter Verwendung eines expressionsoptimierten 4-Komponenten PFV-Vektorsystems, wie in Beispiel 1 beschrieben, hergestellt. Dabei wurden unterschiedliche Mengen der 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
Transfervektoren pcziEGgP (CMV) und pSFFVU3 EGqP (SFFV) eingesetzt. Nach 48 Stunden wurden die zellfreien viralen Überstände geerntet und jeweils 1 ml der verschiedenen unverdünnten Vektorüberstände eingesetzt, um HT1080-Zielzellen mit den im Überstand enthaltenen Vektorpartikeln zu transduzieren. Mit Hilfe von quantitativer PCR wurde die Menge an Zell-RNA nach Extraktion der Gesamt-RNA aus den transfizierten 293T-Zellen sowie die Menge an Virus-RNA nach Extraktion der RNA aus den durch Ultrazentrifugation des Überstandes (10 ml) pelletierten viralen Partikeln bestimmt. Die EGFP-Transgenexpression in den Zielzellen wurde 48 Stunden nach Infektion mittels Durchflusszytometrie gemessen. Es zeigte sich, dass eine Proportionalität zwischen der zur Transfektion der 293T-Zellen 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
eingesetzten DNA-Menge des Transfervektors und der Menge an Zell-RNA, an Virus-RNA sowie der Stärke der Zielzell-Transgenexpression besteht (Fig. 13).
used amount of DNA of the transfer vector and the amount of cell RNA, viral RNA and the strength of the target cell transgene expression (Figure 13).
Claims
(a1 ) einen rekombinanten Vektor, der eine Expressionssequenz enthält, die für ein Gag Protein eines Foamy Virus codiert, und einen rekombinanten Vektor, der eine Expressionssequenz enthält, die für ein Env Protein eines Foamy Virus codiert; oder (a1) a recombinant vector containing an expression sequence encoding a Gag protein of a foamy virus, and a recombinant vector containing an expression sequence encoding an Env protein of a foamy virus; or
(a2) einen rekombinanten Vektor, der eine Expressionssequenz enthält, die für ein Gag Protein eines Foamy Virus codiert, und eine (a2) a recombinant vector containing an expression sequence encoding a Gag protein of a foamy virus, and a
Expressionssequenz enthält, die für ein Env Protein eines Foamy Virus codiert; und Contains expression sequence that codes for an Env protein of a foamy virus; and
(b) einen Transfervektor, der mindestens eine Expressionskassette zur Aufnahme einer für ein Nicht-Foamy-Virus-Genprodukt codierenden Nukleotidsequenz enthält, (b) a transfer vector which contains at least one expression cassette for receiving a nucleotide sequence coding for a non-foamy virus gene product,
wobei der Transfervektor keine Foamy-Virus-Sequenzen enthält. wherein the transfer vector does not contain any foamy virus sequences.
Vektorsystem nach Anspruch 3, wobei einer oder beide der Vektoren gemäß (a1 ) weiter eine Expressionssequenz enthält, die für ein Pol Protein
eines Foamy Virus codiert, oder wobei der Vektor gemäß (a2) weiter eine Expressionssequenz enthält, die für ein Pol Protein eines Foamy Virus codiert, oder wobei das Vektorsystem weiter (c) einen rekombinanten Vektor umfasst, der eine Expressionssequenz enthält, die für ein Pol Protein eines Foamy Virus codiert. Vector system according to claim 3, wherein one or both of the vectors according to (a1) further contains an expression sequence for a Pol protein a foamy virus, or wherein the vector according to (a2) further contains an expression sequence which codes for a Pol protein of a foamy virus, or wherein the vector system further comprises (c) a recombinant vector which contains an expression sequence which codes for a Pol Protein encoded by a foamy virus.
5. Vektorsystem nach Anspruch 4, wobei die Expressionssequenz für ein nicht-funktionales Pol Protein eines Foamy Virus codiert. 5. Vector system according to claim 4, wherein the expression sequence codes for a non-functional Pol protein of a foamy virus.
6. Vektorsystem nach einem der vorhergehenden Ansprüche, wobei die 6. Vector system according to one of the preceding claims, wherein the
Expressionskassette des Transfervektors einen Promotor umfasst, der aus der Gruppe, bestehend aus SFFV U3, CMV, CAGG und MLV LTR, ausgewählt ist. Expression cassette of the transfer vector comprises a promoter selected from the group consisting of SFFV U3, CMV, CAGG and MLV LTR.
7. Vektorsystem nach einem der vorhergehenden Ansprüche, wobei 7. Vector system according to one of the preceding claims, wherein
mindestens eine der Expressionssequenzen zur Expression in Homo sapiens optimiert ist. at least one of the expression sequences is optimized for expression in Homo sapiens.
8. Vektorsystem nach einem der Ansprüche 3 bis 7, wobei mindestens einer der rekombinanten Vektoren gemäß (a1 ) oder der rekombinante Vektor gemäß (a2) und/oder ggf. der rekombinante Vektor gemäß (c) eine Expressionssequenz enthält/enthalten, die für eine nicht-funktionale Foamy Virus Integrase und/oder eine nicht-funktionale Foamy-Virus- Reverse-Transkriptase und/oder eine nicht-funktionale Foamy-Virus- Protease codiert. 8. Vector system according to one of claims 3 to 7, wherein at least one of the recombinant vectors according to (a1) or the recombinant vector according to (a2) and / or optionally the recombinant vector according to (c) contains / contain an expression sequence which is for a non-functional foamy virus integrase and/or a non-functional foamy virus reverse transcriptase and/or a non-functional foamy virus protease.
9. Vektorsystem nach einem der vorhergehenden Ansprüche, wobei der Transfervektor eine Nukleotidsequenz aufweist, die aus der Gruppe bestehend aus SEQ ID NO: 1 und SEQ ID NO: 2 ausgewählt ist. 9. Vector system according to one of the preceding claims, wherein the transfer vector has a nucleotide sequence which is selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2.
10. Vektorsystem nach einem der vorhergehenden Ansprüche, wobei der Transfervektor eine in die Expressionskassette funktional eingefügte Nukleotidsequenz enthält, die für mindestens ein in Bezug auf das 10. Vector system according to one of the preceding claims, wherein the transfer vector contains a nucleotide sequence functionally inserted into the expression cassette, which is for at least one in relation to
Retrovirus heterologes Genprodukt codiert.
Retrovirus heterologous gene product encoded.
1 1 . Vektorsystem nach Anspruch 10, wobei das Genprodukt aus der Gruppe, bestehend aus Peptiden, Proteinen, siRNAs, shRNAs, miRNAs, pri- miRNAs, pre-miRNAs und Antisense-RNAs, ausgewählt ist. 1 1 . Vector system according to claim 10, wherein the gene product is selected from the group consisting of peptides, proteins, siRNAs, shRNAs, miRNAs, pri-miRNAs, pre-miRNAs and antisense RNAs.
12. Vektorsystem nach Anspruch 1 1 , wobei die Nukleotidsequenz für ein Reporterprotein oder -peptid codiert. 12. Vector system according to claim 1 1, wherein the nucleotide sequence codes for a reporter protein or peptide.
Wirtszelle, die mit einem Vektorsystem nach einem der vorhergehenden Ansprüche transfiziert ist. Host cell transfected with a vector system according to one of the preceding claims.
14. Wirtszelle nach Anspruch 13, die zur Herstellung und Ausschleusung von Retrovirus-Partikeln durch Expression mindestens der essentiellen Virus- Proteine über den/die Vektor(en) gemäß (a) ausgelegt ist. 14. Host cell according to claim 13, which is designed for the production and delivery of retrovirus particles by expressing at least the essential virus proteins via the vector(s) according to (a).
15. Verfahren zur Herstellung der Wirtszelle nach Anspruch 13 oder 14, 15. Method for producing the host cell according to claim 13 or 14,
umfassend das Transfizieren einer geeigneten Zelle mit dem Vektorsystem nach einem der Ansprüche 1 bis 12. comprising transfecting a suitable cell with the vector system according to any one of claims 1 to 12.
Kit zur Expression eines nicht-retroviralen Genprodukts, umfassendKit for expressing a non-retroviral gene product, comprising
(i) ein Vektorsystem nach einem der Ansprüche 1 bis 12 und (i) a vector system according to one of claims 1 to 12 and
(ii) Zellen, die als Wirtszellen gemäß Anspruch 14 geeignet sind. (ii) Cells suitable as host cells according to claim 14.
Kit nach Anspruch 16, weiter umfassend The kit of claim 16, further comprising
(iii) Zellen, die zur Infektion mit Retrovirus-Partikeln geeignet sind. (iii) Cells suitable for infection with retrovirus particles.
Verfahren zur Expression eines nicht-retroviralen Genprodukts in einer Zelle, umfassend die Schritte: Method for expressing a non-retroviral gene product in a cell, comprising the steps:
(A) Transfizieren einer Zelle mit einem Vektorsystem nach einem der Ansprüche 10 bis 12, wobei die Zelle zur Herstellung und Ausschleusung von Retrovirus-Partikeln durch Expression mindestens der essentiellen Virus-Proteine über den/die Vektor(en) gemäß (a) ausgelegt ist,
(B) Kultivieren der transfizierten Zelle in einem Kulturmedium unter Bedingungen, die die Bildung und Ausschleusung von Retrovirus- Partikeln in das Kulturmedium erlauben, (A) Transfecting a cell with a vector system according to one of claims 10 to 12, wherein the cell is designed for the production and discharge of retrovirus particles by expressing at least the essential virus proteins via the vector(s) according to (a). , (B) cultivating the transfected cell in a culture medium under conditions that allow the formation and release of retrovirus particles into the culture medium,
(C) Gewinnen eines Retrovirus-Partikel enthaltenden Überstands aus der Kultur von Schritt (B) und (C) Obtaining a supernatant containing retrovirus particles from the culture of steps (B) and
(D) Transduzieren von Zellen, die mit den Retrovirus-Partikeln infizierbar sind, mit dem ggf. verdünnten Kulturüberstand von Schritt (C). (D) Transducing cells that can be infected with the retrovirus particles with the optionally diluted culture supernatant from step (C).
Verfahren zur Expression eines nicht-retroviralen Genprodukts in einem Wirtsorganismus, umfassend das Einbringen einer Wirtszelle nach Method for expressing a non-retroviral gene product in a host organism, comprising introducing a host cell
Anspruch 14 in den Wirtsorganismus, wobei ein oder mehrere Zelle(n) des Wirtsorganismus durch Retrovirus-Partikel infizierbar ist/sind.
Claim 14 in the host organism, wherein one or more cells of the host organism can be infected by retrovirus particles.
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