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WO1998038322A1 - Produits de recombinaison d'acides nucleiques servant a exprimer des transgenes de maniere durable - Google Patents

Produits de recombinaison d'acides nucleiques servant a exprimer des transgenes de maniere durable Download PDF

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Publication number
WO1998038322A1
WO1998038322A1 PCT/EP1998/000992 EP9800992W WO9838322A1 WO 1998038322 A1 WO1998038322 A1 WO 1998038322A1 EP 9800992 W EP9800992 W EP 9800992W WO 9838322 A1 WO9838322 A1 WO 9838322A1
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Prior art keywords
nucleic acid
acid construct
transcription factor
construct according
binding site
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PCT/EP1998/000992
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German (de)
English (en)
Inventor
Hendrik Veelken
Albrecht Lindemann
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KLINIKUM DER ALBERT-LUDWIGS-UNIVERSITäT FREIBURG
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Priority to AU66227/98A priority Critical patent/AU6622798A/en
Publication of WO1998038322A1 publication Critical patent/WO1998038322A1/fr

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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
    • C12N15/635—Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline
    • 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
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/005—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination repressible enhancer/promoter combination, e.g. KRAB
    • C12N2830/006—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination repressible enhancer/promoter combination, e.g. KRAB tet repressible
    • 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
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES

Definitions

  • the nucleic acid constructs according to the invention are used for the expression of transgenes in host cells. Genetic engineering makes it possible to transfer certain genes from a donor into recipient cells. This transferred gene (transgene) is then expressed in the host cell. In higher organisms (eukaryotes) in particular, regulation of expression involves considerable difficulties.
  • the present invention relates to nucleic acid constructs, in particular vectors, which enable permanent, long-term expression of transgenes in transformed cells.
  • Hofmann et al. (Proc. Natl. Acad. Sci. USA, Vol. 93 ( May 1996), pp. 5185-5190) describe an autoregulatory cassette which enables the reversible induction of transgene expression as a function of tetracycline.
  • the Hofmann et al. The nucleic acid construct described uses a minimal promoter derived from the cytomegalovirus as the promoter. In its natural form, this promoter controls the expression of the very early proteins of cytomegalovirus (CMV Immediate Early Minimal Promoter).
  • the present invention therefore relates to nucleic acid constructs which are suitable for expressing a foreign gene in a host cell and which have the following components:
  • nucleic acid construct is understood to mean an arrangement of various components which consist of nucleic acid, in particular DNA.
  • the components of the nucleic acid constructs according to the invention act functionally on one another and enable the expression of a transgene under controlled conditions.
  • the individual components of the nucleic acid constructs according to the invention can preferably be located on one nucleic acid construct or they can be distributed over two or more constructs. However, the prerequisite is that in the latter case the individual nucleic acid constructs are in one cell.
  • the nucleic acid constructs according to the invention are used to express a foreign gene in a host cell.
  • the term “foreign gene” or “transgene” is understood to mean a gene which codes for a gene product which, after its expression, causes certain effects.
  • the transgenes are preferably genes which code for cytokines.
  • the term cytokines encompasses a group of immunomodulatory proteins, which are also called immunotransmitters.
  • the cytokines act as humoral regulators that regulate the functional activities of certain cells.
  • the term "cytokines" includes the interleukins, interferons and colony stimulating factors.
  • the colony-stimulating factors in particular G-CSF (granolocyte colony-stimulating factor), are particularly preferred.
  • expression is increased by incorporating at least one binding site for a transcription factor at a suitable location in the promoter.
  • the gene coding for the transcription factor is arranged downstream of the promoter and is controlled by it.
  • the binding site for the transcription factor which is incorporated into the promoter according to the invention, preferably originates from a different gene arrangement and therefore does not occur in the corresponding naturally occurring promoter.
  • the nucleic acid constructs according to the invention contain, as a special feature within a constitutively active promoter, a binding sequence for a transcription factor and the gene of the same transcription factor at the 3 'end of the construct.
  • the translation of the gene of this transcription factor is made possible by suitable fusion with the "internal ribosome entry sequence" of the encephalomyocarditis virus.
  • the expression of the transgene and the transcription factor is controlled by the same promoter.
  • Such a construct is therefore also called "dicistronic".
  • the expression of the transcription factor has a feedback-enhancing effect on its own expression and on the expression of the transgene in the arrangement according to the invention.
  • the constitutively active function of the promoter is significantly enhanced by this mechanism.
  • nucleic acid constructs according to the invention can be regulated in terms of expression.
  • nucleic acid constructs have the following components:
  • transgene at least one transgene and a chimeric gene coding for a repressor, which can bind to the binding site for a repressor (b), fuses with at least part of a transactivation factor.
  • a binding site for a repressor is then coupled to the functional part of the promoter.
  • This binding site can preferably be located within the promoter.
  • the nucleic acid construct contains a chimeric gene that codes for a repressor that can bind to the binding site for the repressor.
  • the chimeric gene also contains a gene for a transactivation factor.
  • the chimeric gene product binds to the substrate, thereby preventing binding to the repressor binding site.
  • the repressor binds to the binding site for the repressor.
  • the increase in expression is then brought about by the other part of the chimeric gene product, namely the transactivation factor.
  • a preferred regulation system is the tetracycline repressor system, which originates from the transposon 10 from E. coli.
  • the regulatory substrate is tetracycline. If tetracycline is present in the cell, the repressor and tetracycline bind and the chimeric gene product cannot bind to the tet operator. This does not increase transcription. If there is no tetracycline in the cell, the product of the chimeric gene can bind to the tet operator and an increase in transcription, which leads to an increase in expression, takes place via the VP16 protein of the herpes simplex virus.
  • the binding site for the repressor (b) is tet operator, which is present several times in a tandem-like arrangement in a particularly preferred embodiment.
  • an internal ribosome entry sequence is arranged between the transgene (c) and the gene coding for the transcription factor or the chimeric gene (d), the IRES of the encephalomyocarditis virus being particularly preferred.
  • the gene coding for a transcription factor is a corresponding human-derived gene.
  • the gene for GATA-1 can be named as a transcription factor that binds to the binding sequence (b) GATA. This gene is from Evans et al. in Mol. Cell. Biol., 11 (1991) pp. 843-853.
  • Another suitable transcription factor is called HNF3 and is described by Colrichter et al. in Blood 84 (1994), pp. 3394-3404. This transcription factor binds to the binding sequence
  • This gene for the transcription factor can also be part of a chimeric gene, the other part of the chimeric gene coding for a repressor that can bind to the binding site (b).
  • a repressor that can bind to the binding site (b).
  • An example of this repressor would be the tetracycline repressor of transposon 10 from E. coli or another repressor that can be regulated by a drug, preferably an antibiotic.
  • the binding site (b) in the nucleic acid construct must then be designed so that the transcription factor or the product of the chimeric gene can bind to the binding site.
  • the nucleic acid constructs have a host cell-related, constitutively active promoter ("housekeeping promoter"), which is either from the host itself or a closely related species.
  • the host cell-related promoter is preferably the human ⁇ -actin promoter.
  • the part of the chimeric gene (d) which codes for a repressor which can bind to the binding site (b) is the tetracycline repressor of the transposon 10 from E. coli.
  • the part of a gene which codes for a transactivation factor, such as, for example, the VP16 protein of the herpes simplex virus, is preferably fused to this.
  • the individual components (a), (b), (c) and (d) are arranged on the polynucleotide in the 5 'to 3' direction.
  • the nucleic acid constructs according to the invention are generally bare transfectable DNA or plasmid vectors. It is particularly advantageous with the nucleic acid constructs according to the invention that expression can be achieved in the host cells, even if the constructs are not in the form of a viral, in particular retroviral, vector. The use of viral vectors is less preferred in gene therapy due to the unpredictable risks.
  • vectors or corresponding nucleic acid constructs can be used without
  • Origin of replication for the transformation of a eukaryotic host cell in particular of human cells such as blood stem cells.
  • FIG. 1A schematically shows monocistronic G-CSF expression constructs. All plasmids are based on pBluescript. ß-Actin is the promoter of the human ß-actin gene. CMV is the immediate-early promoter of CMV (cytomegalovirus). G-CSF is human G-CSF cDNA. TATA is the TATA box. polyA means the small intron and polyadenylation signal of the SV40 virus.
  • tetO means tande -tet operators.
  • the constructs pßACPl.GCSF, pßACP2.GCSF and pßACX.GCSF were generated by inserting tetO trimers at the indicated positions in relation to the TATA box in pßAC.GCSF.
  • FIG. 1B represents a comparison of the insertion sites of the tet operator sequences within the human ⁇ -actin promoter.
  • the tetO trimers were used at the restriction sites indicated in FIG. 1A.
  • the corresponding expression constructs were transiently expressed in KMST-6 cells. G-CSF concentrations in the supernatants were measured 48 hours after transfection by ELISA.
  • FIG. 2 shows a schematic representation of the dicistronic constructs. All plasmids are based on pBluescript. The abbreviations have the meaning given above.
  • IRES Internal Ribosome Entry Sequence (of the Encephalomyocarditis Virus) and tetR / VP16 means chimeric transcription activator with the tet repressor and VP16 activity.
  • Figure 3 shows the kinetics of tetracycline-induced activation and repression of G-CSF production in Balb 3T3 clones that were stably transfected with ptetOtata.GCSF.iresTTAS (one clone) and pßACP2.GCSF. iresTTA
  • FIG. 4 shows Table 1, which enables a comparison of the different promoters in the case of transient expression of G-CSF in KMST-6 cells.
  • An expression plasmid for tetR / VP16 (pUHD 15-1) or a control plasmid (pSP65) was co-transfected by cationic lipofection with the indicated G-CSF plasmid in a 1: 1 (weight / weight) ratio.
  • the G-CSF concentrations in the culture supernatants were measured in the absence or presence of 0.1 ⁇ g / ml tetracycline 48 hours after transfection with ELISA. The values given correspond to average values of new determinations in ng / ml ( ⁇ standard deviation).
  • FIG. 5 shows Table 2, which enables a comparison of the different constructs in the case of transient expression of G-CSF in KMST-6 cells.
  • Equimolar amounts of the plasmids were transfected by cationic lipofection.
  • Appropriate amounts of the control plasmid (pSP65) were added to pßAC.GCSF and pCMV.GCSF to ensure that appropriate amounts of DNA were present in each transfection complex.
  • the G-CSF concentrations in the culture supernatants in the absence or presence of 0.1 ⁇ g / ml tetracycline were measured 48 hours after transfection using the ELISA technique. The values correspond to average values of new determinations in ng / ml ( ⁇ standard deviation).
  • FIG. 6 shows Table 3, which enables a comparison of the different constructions for the stable expression of G-CSF in Balb3T3 cells.
  • An expression plasmid for Neo r (placOSTHNeo) was co-transfected with the indicated G-CSF plasmid in one by cationic lipofection 1: 9 ratio.
  • Stably transfected cells were selected by adding 1 mg / ml G418 to the culture medium. Individual colonies were isolated and expanded.
  • G-CSF concentrations of seven independent clones for each G-CSF expression plasmid were measured in defined culture supernatants in the absence or presence of 0.1 ⁇ g / ml tetracycline. Set the values
  • FIG. 7 shows a comparison of the expression of a vector according to the invention (represented by filled circles) in relation to a corresponding comparison construct (open circles).
  • Recombinant human G-CSF is expressed in mice by the nucleic acid construct according to the invention and the extent of expression is measured by the increase in leukocytes.
  • the human ß-actin promoter was taken from a plasmid which contained the ß-actin promoter in a 2.9 kb Sal I - Sac I fragment.
  • the cDNA encoding G-CSF was obtained by RT-PCR amplification of RNA extracted from lipopolysaccharide-stimulated peripheral blood mononuclear cells (plasmids pUHD 15-1 and pUHC 13-3 were prepared according to Gossen and Bujard (PNAS [1992 ], Pp. 5547-5551)
  • the IRES sequence of encephalomyocarditis virus is described, for example, in Zimmermann et al. (Virology [1994], pp. 366-372).
  • the Balb3T3 fibroblast mice were obtained from the ATCC.
  • the immortalized human fibroblast cell lines KMST-6 was developed by Namba et al. (Int. J.Cancer [1985], pp. 275-280).
  • the cell lines were cultured in Dulbecco's modified high glucose Eagle's medium supplemented with 10% fetal calf serum, 2 mM glutamine, 2 mM sodium pyruvate and 50 ⁇ g / ml gentamycin.
  • the plasmids for the transfections were purified by anion exchange chromatography. Remaining contamination with endotoxin was removed by treatment with polymyxin B.
  • the determination of the G-CSF concentrations in the culture supernatants was carried out using a commercially available ELI ⁇ A test. In transient expression experiments, the supernatants were collected 48 hours after transfection. In order to generate defined supernatants of the stably transfected clones, 2.5 ⁇ 10 ⁇
  • the plasmid pCMV.GCSF shown in FIG. 1A, is based on pBluescript II KS (Stratagene, La Jolla, CA) and contains the human G-CSF cDNA under the control of the immediate / early promoter of CMV and a polyadenylation signal which comes from SV40 .
  • the CMV promoter control was replaced by the human ⁇ -actin promoter (according to the invention) or the minimal promoter with tetO in order to obtain pßAC.GCSF and ptetOtata.GCSF.
  • the 11th ATG codon of the EMCV IRES from pSport / PV / 2/5 '- Aat was fused to the translation initiation codon of the tetR / VP16 cDNA from pUHD 15-1 using a two-step PCR technology. All fragments generated using PCR technology were checked by DNA sequencing.
  • Tandem tetO sequences were obtained by PCR amplification of the tetO heptamer from the plasmid pUHC 13-3 with tetO-specific primers, Pst I or Xho I restriction sites being added at both ends of the PCR products.
  • the polyacrylamide gel electrophoresis of the PCR products showed a ladder of tetO concatamers.
  • tetO trimers were excised from the gel and inserted at the Pst I sites at position -423 and -1663 with respect to the TATA box or at an Xho I site at position -23. These modified promoters were then cloned into pßAC.GCSF instead of the unmodified ⁇ -actin promoter. This is shown in Figure 1A.
  • the transient expression of these plasmids in KMST- 6 cells identified the insertion of the tetO trimer at the Pst I site at position -423 (pßACP2.GCSF) as an arrangement that retained 85% of the basic promoter activity.
  • FIG. 1B Insertions at other sites or the use of tetO-hexamers led to an at least 65% reduction in promoter activity (FIG. 1B).
  • G-CSF expression levels of pßAC.GCSF corresponded approximately to the level when either pUHD 15-1 or a control plasmid (pSP65) were co-transfected, regardless of the presence or absence of tetracycline (tet) in that Culture medium. This is shown in Table 1 of Figure 4.
  • This construct corresponds to ptetOtata.GCSF, shown in FIG. 1A.
  • the results confirm the function of the tetO sequences within the ß-actin promoter and clearly show the ability of the tetR / VP16 system of transcriptional regulation in the cell lines used according to the invention. However, all of these promoters resulted in a significantly lower expression compared to the strong immediate-early CMV promoter in pCMV.GCSF. Corresponding results were obtained in Balb3T3 cells.
  • dicistronic expression plasmids were constructed for the simultaneous expression of a therapeutic gene and tetR / VP16.
  • the translation initiation codon of the tetR / VP16 cDNA was fused to the 11th ATG codon from EMCV in a two-step PCR procedure and the IRES-tetR / VP16 sequence obtained was inserted downstream of the G-CSF cDNA in pßAC.GCSF , pßACP2.GCSF and ptetOtata.GCSF. This is shown in Figure 2.
  • the dicistronic vector was compared to the monocistronic G-CSF expression plasmid in transient expression experiments in KMST-6 cells.
  • pCMV.GCSF and pßAC.GCSF were mixed with appropriate amounts of pSP65 to maintain equimolar ratios of the expression vector within the 2 ⁇ g DNA used for standard transfection complexes.
  • the CMV promoter was about three times stronger than the wild-type ⁇ -actin promoter. This is shown in Table 2 of Figure 5.
  • the feedback transcription activator arrangement with the tetO-modified ⁇ -actin promoter pßACP2.GCSF.
  • IresTTA led to even higher expression levels compared to the strong CMV promoter.
  • the transcription of pßACP2.GCSF. iresTTA only partially inhibited by the addition of tetracycline.
  • the expression levels of the feedback activating vector were also examined after integration into the cellular genome.
  • Balb3T3 cells were co-transfected with one of the dicistronic Plasmids and placOSTHNeo. Individual neoycin-resistant colonies were isolated and expanded and defined supernatants were examined for the production of G-CSF. This is shown in Table 3 of Figure 6.
  • the tetO - ires- tetR / VP16 arrangement resulted in an average three-fold increase in G-CSF production compared to the conventional ß-actin promoter (p ⁇ 0.001).
  • mice were Balb3T3 cells either with the expression vector pßACP2tetO according to the invention.
  • GCSF. irestTA or with the comparison vector pßAC.GCSF (without a binding site for a transcription factor and without a gene coding for a transcription factor), each in combination with an expression construct for neomycin phosphotransferase transfected by cationic lipofection.
  • Stable transfected clones were isolated by selection with G418 and then expanded.
  • One Balb3T3 clone of each plasmid was selected for in vivo experiments.
  • Balb3T3 clone produces approximately 1.2 ⁇ g of recombinant human G-CSF per 24 h and 10 ⁇ cells.
  • the Balb3T3 clone transfected with the reference construct (pßAC.GCSF) produces approximately 0.25 ⁇ g of recombinant human
  • mice 5 10 ⁇ cell SCID-type mice were injected subcutaneously from each of the two clones. Per plasmid or clone 3 mice each used. The leukocyte count of the mice was then determined as a measure of the expression of the therapeutic genes.

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Abstract

Les produits de recombinaison d'acides nucléiques décrits permettent d'exprimer un gène étranger dans une cellule hôte et comprennent les constituants suivants: (a) au moins une partie d'un promoteur qui correspond à un promoteur constitutif actif apparenté à l'hôte; (b) au moins un site de liaison d'un facteur de transcription; (c) au moins un transgène; et (d) au moins un gène de codage d'un facteur de transcription qui peut se lier au site de liaison d'un facteur de transcription (b).
PCT/EP1998/000992 1997-02-25 1998-02-20 Produits de recombinaison d'acides nucleiques servant a exprimer des transgenes de maniere durable WO1998038322A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU66227/98A AU6622798A (en) 1997-02-25 1998-02-20 Nucleic acid constructs for durable transgene expression

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DE19707493.6 1997-02-25
DE1997107493 DE19707493C1 (de) 1997-02-25 1997-02-25 Nucleinsäurekonstrukte zur lang andauernden Expression von Transgenen

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118291541A (zh) * 2024-06-05 2024-07-05 上海凌医生物科技有限公司 一种诱导型启动子

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20230052011A1 (en) * 2019-12-04 2023-02-16 Crispr Therapeutics Ag Regulatable expression systems

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994029442A2 (fr) * 1993-06-14 1994-12-22 Basf Aktiengesellschaft Maitrise precise de l'expression des genes dans les cellules encaryotes au moyen de promoteurs reagissant a la tetracycline
WO1996001313A1 (fr) * 1994-07-01 1996-01-18 Hermann Bujard Modulateurs de transcription regules par la tetracycline
WO1996033272A1 (fr) * 1995-04-18 1996-10-24 KLINIKUM DER ALBERT-LUDWIGS-UNIVERSITäT FREIBURG Vecteurs de transfection de cellules eucaryotes, leur utilisation et cellules cibles transfectees a l'aide desdits vecteurs

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994029442A2 (fr) * 1993-06-14 1994-12-22 Basf Aktiengesellschaft Maitrise precise de l'expression des genes dans les cellules encaryotes au moyen de promoteurs reagissant a la tetracycline
WO1996001313A1 (fr) * 1994-07-01 1996-01-18 Hermann Bujard Modulateurs de transcription regules par la tetracycline
WO1996033272A1 (fr) * 1995-04-18 1996-10-24 KLINIKUM DER ALBERT-LUDWIGS-UNIVERSITäT FREIBURG Vecteurs de transfection de cellules eucaryotes, leur utilisation et cellules cibles transfectees a l'aide desdits vecteurs

Non-Patent Citations (1)

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Title
HOFMANN, A. ET AL.: "Rapid retroviral delivery of tetracycline-inducible genes in a single autoregulatory cassette", PROC. NATL. ACAD. SCI. USA, vol. 93, 1996, pages 5185 - 5190, XP002070633 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118291541A (zh) * 2024-06-05 2024-07-05 上海凌医生物科技有限公司 一种诱导型启动子

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