WO1997041243A2 - Systeme d'expression cytoplasmique de gene mettant en application un autogene procaryote d'arn polymerase - Google Patents

Systeme d'expression cytoplasmique de gene mettant en application un autogene procaryote d'arn polymerase Download PDF

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WO1997041243A2
WO1997041243A2 PCT/US1997/007030 US9707030W WO9741243A2 WO 1997041243 A2 WO1997041243 A2 WO 1997041243A2 US 9707030 W US9707030 W US 9707030W WO 9741243 A2 WO9741243 A2 WO 9741243A2
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sequence
cat
promoter
cells
rna polymerase
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PCT/US1997/007030
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WO1997041243A3 (fr
WO1997041243A9 (fr
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Leaf Huang
Paul Robbins
Daniel Jaffurs
Marni Brisson
Song Li
Jingping Yang
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University Of Pittsburgh
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Priority to JP09539094A priority patent/JP2000510334A/ja
Priority to EP97921403A priority patent/EP0910653A2/fr
Publication of WO1997041243A2 publication Critical patent/WO1997041243A2/fr
Publication of WO1997041243A9 publication Critical patent/WO1997041243A9/fr
Publication of WO1997041243A3 publication Critical patent/WO1997041243A3/fr

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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/108—Plasmid DNA episomal vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • 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
    • C12N2840/206—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES having multiple IRES

Definitions

  • the present invention relates to the expression of nucleic acid sequences in eukaryotic cells. More specifically, the invention relates to a cytoplasmic expression system which utilizes prokaryotic RNA polymerase autogenes to facilitate the cytoplasmic expression of DNA in cells.
  • cytoplasmic uptake of DNA by eukaryotic cells is more efficient and prokaryotic RNA polymerases reside only in the cytoplasm of eukaryotic cells, due to the absence of a nuclear localization signal in the molecules.
  • cytoplasmic expression systems have drawbacks since high, continuous levels of expression of DNA in cells via such systems is dependent on a continuous supply of prokaryotic RNA polymerase in the cytoplasm of the cells.
  • T7 expression vectors must be codelivered to cells with an exogenous supply of T7 RNA polymerase or utilized in cell lines which express T7 RNA polymerase.
  • T7 expression vectors in eukaryotic cells since the T7 RNA polymerase gene is a bacterial gene not expressed endogenously in eukaryotic cells.
  • investigators have attempted to design T7 RNA polymerase autogenes which would function as a self-initiating and self-amplifying source of T7 RNA polymerase in the cytoplasm of eukaryotic cells.
  • Gao et al. ((1993) Nucleic Acids Res. 21: 2867- 2872) described a T7 autogene designated pT7AUT02C , that successfully supported cytoplasmic expression in eukaryotic cells of a reporter gene driven by the bacteriophage T7 promoter.
  • this autogene was able to produce T7 RNA polymerase only when codelivered to cells with an amount of exogenous T7 RNA polymerase sufficient to initiate the autocatalytic production of T7 RNA polymerase from the T7 promoter of pT7AUT02C.
  • the pT7AUT02C " autogene was not truly a self-generating source of T7 polymerase in that it required delivery of exogenous T7 polymerase to start the autocatalytic cycle.
  • Deng et al. (1993) Gene , 143:245-249) refer to an autogene plasmid which contains the T7 promoter, the encephalomyocarditis internal ribosomal entry sequence (EMC IRES) and the T7 RNA polymerase gene, in vitro transcripts of this autogene alone, or in combination with the autogene plasmid DNA, were reported to support foreign o gene expression in vitro.
  • EMC IRES encephalomyocarditis internal ribosomal entry sequence
  • WO 94/26911 refers to a construct which contains a T7 RNA polymerase gene, an EMC IRES and a T7 promoter. Incubation of this construct with T7 RNA polymerase is reported to result in binding of the T7 RNA polymerase to 0 the construct prior to the introduction of the construct into cells thereby permitting the T7 RNA polymerase: construct complex to serve as a self-initiating and self- amplifying source of T7 RNA polymerase upon entry into the cytoplasm of cells.
  • T7 RNA polymerase autogene that can yield continuous, high level expression of T7 RNA polymerase in eukaryotic cells without the need for exogenous T7 polymerase enzyme to initiate the amplification process.
  • Such autogenes would be useful for both in vitro and in vivo protein expression, including use in gene therapy.
  • Gene therapy has become one of the fastest developing fields of biomedicine in recent years (Anderson, W.F. (1992) Science. 256: 808-813).
  • the prokaryotic RNA polymerase autogene of the invention is a DNA construct which includes at a minimum, the following elements operatively linked in 5' to 3' order: a eukaryotic promoter, a cognate promoter of the prokaryotic RNA polymerase and a nucleic acid sequence encoding the a prokaryotic RNA polymerase.
  • the cognate promoter in the autogene construct may be separated from the RNA polymerase gene by an internal ribosome entry site sequence (IRES) .
  • the invention further relates to the use of this autogene in cytoplasmic expression systems in vitro and $ in vivo to express target sequences.
  • the cytoplasmic expression system is a "dual sequence cytoplasmic expression system" which contains a first sequence comprising the prokaryotic RNA polymerase autogene and a second sequence comprising 0 the cognate RNA polymerase promoter operatively linked to a target sequence.
  • the cytoplasmic expression system may be a "single sequence cytoplasmic expression system" which consists of a single nucleic acid 5 sequence containing in 5' to 3' order: a eukaryotic promoter, a cognate promoter of the prokaryotic RNA polymerase, a nucleic acid sequence encoding the RNA polymerase, a second copy of the cognate promoter and a target sequence.
  • the cognate promoter is separated from the target sequence in the dual and single sequence systems by an IRES.
  • the invention further provides pharmaceutical compositions comprising either the dual or single sequence cytoplasmic expression systems.
  • the compositions of the invention may be administered prophylactically and/or therapeutically.
  • the invention also relates to a kit comprising the prokaryotic RNA polymerase autogene alone, in combination with a second sequence containing the cognate RNA polymerase promoter operatively linked to a target sequence as a dual sequence cytoplasmic expression system, or as a single sequence in combination with a second copy of the cognate RNA polymerase promoter operatively linked to a target sequence.
  • the invention also provides cell lines stably transformed with the prokaryotic RNA polymerase autogene of the invention.
  • FIG. 1 shows maps of the linear DNA fragments and conventional plasmids used in the transfection studies. Sequences of primers used to generate the linear PCR fragments are also shown. The abbreviations for the DNA sequences are as follows: pT7-CAT: T7-CAT plasmid; fT7-CAT: T7-CAT PCR fragment; pUCCMV-CAT: CMV-CAT plasmid; fCMV-CAT: CMV-CAT PCR fragment; pCMV/T7-T7pol: the T7 autogene plasmid where the T7 RNA polymerase gene is under the control of both CMV and T7 promoters; pT7: T7 promoter; tT7: T7 terminator; T7pol: T7 RNA polymerase gene; IRES: internal ribosome entry site sequence of encephalomyocarditis virus.
  • Figure 2 shows plasmid maps for pcDNA3, pAR3126 and pCMV/T7-T7pol.
  • a Hindlll/BamHI fragment from pAR3126 containing the cDNA of T7 RNA polymerase was inserted into the corresponding sites of the pcDNA3 plasmid vector to generate the pCMV/T7-T7pol autogene.
  • FIG. 3 shows a plasmid map for T7 RNA polymerase autogene designated PAUTOM2-C which comprises in 5'-3' order: a CMV immediate early promoter; a mutant T7 promoter (m2 Promoter) an Encv IRES, a T7 RNA polymerase gene and a T7 terminator.
  • Figure 4 shows a plasmid map for a T7 RNA polymerase autogene designated PT7-H1-AUTOD which comprises in 5' to 3' order: a CMV immediate early promoter, a T7 promoter fused to the lac operator, an Emcv IRES, a T7 RNA polymerase gene and a T7 terminator'.
  • Figure 5 shows a schematic of a single sequence cytoplasmic expression system which contains in a single nucleic acid sequence the following elements in 5' to 3' order: a CMV promoter, a T7 promoter, a T7 RNA polymerase gene, a second copy of the T7 promoter, an EMCV IRES and a CAT reporter gene.
  • Figure 6 shows a schematic of a single sequence cytoplasmic expression system designated pTex 2 which contains in a single nucleic acid sequence, the following elements in 5' to 3' order: a CMV immediate early promoter, a mutant T7 promoter (m2) , an IRES, T7 RNA polymerase gene, a wild-type T7 promoter (T7 WT) , an IRES and an IRAP reporter gene.
  • pTex 2 which contains in a single nucleic acid sequence, the following elements in 5' to 3' order: a CMV immediate early promoter, a mutant T7 promoter (m2) , an IRES, T7 RNA polymerase gene, a wild-type T7 promoter (T7 WT) , an IRES and an IRAP reporter gene.
  • Figure 7 shows CAT activity as a function of pT7-CAT and fT7-CAT DNA concentration.
  • CAT assays were performed 48 hours following the transfection.
  • Figure 8 shows CAT expression as a function of time. 0.27 pmol of pT7-CAT or fT7-CAT DNA was delivered to 293-T7 cells by DC-chol:DOPE liposomes as described in Figure 4. Cells were collected daily for 9 days and split 1:1 once on day 4 post-transfection when they were 100% confluent.
  • Figure 9 shows CAT activity 48 hours following cotransfection of normal 293 cells for 4 hours with a mixture of 0.27 pmol pT7-CAT or fT7CAT and varying mole amounts of pCMV/T7-T7pol autogene complexed with DC- cholrDOPE liposomes (10 nmol lipid/ ⁇ g DNA).
  • Figure 10 shows CAT activity in normal 293 cells as a function of pT7-CAT and fT7-CAT DNA concentration.
  • 293 cells were transfected for 4 hours with various amounts of pT7-CAT or fT7-CAT codelivered with pCMV/T7- T7pol (5:1 mol/mol of pT7-CAT or fT7-CAT to pCMV/T7-T7 pol) by DC-chol:DOPE liposomes (10 nmol lipid/ ⁇ g DNA).
  • Figures IIA and 11B show CAT expression ( Figure IIA) and toxicity ( Figure 11B) as a function of time in 293 cells transfected for four hours with 0.27 pmol pT7- CAT or fT7-CAT and 54.11 fmol pCMV/T7-T7pol autogene complexed with DC-Choi: DOPE liposomes (10 nmol lipid/ ⁇ g DNA) .
  • Figure IIA CAT activity was determined 48 hours after transfection and in Figure HB, toxicity was measured as protein recovered in transfected cells 48 hours after transfection.
  • Figures 12A and 12B show transfection efficiency (Figure 12A) and toxicity (Figure 12B) of 293 cells co- transfected for four hours with pT7-CAT (1 ⁇ g) and T7 RNA polymerase (150 units) and/or T7 autogenes (0.3 ⁇ g) pT7AUT02C- or pCMV/T7-T7 pol complexed with 10 nmol of DC- chol:DOPE liposomes (3:2, mol/mol).
  • transfection efficiency was measured as CAT activity determined 48 hours after transfection and in Figure 12B, toxicity was measured as protein recovered in transfected cells 48 hours after transfection as a percentage of protein recovered in untreated control cells.
  • Figures 13A and 13B show a comparison of two T7 autogenes, pT7 AUTO 2C " and pCMV/T7-T7pol, for their ability to sustain pT7-CAT expression over time (Figure 13A) and their relative toxicities over time (Figure 13B)
  • pT7 AUTO 2C * 0.3 ⁇ g
  • 150 units of T7 RNA polymerase complexed with 10 nmol of DC- Chol:D0PE (3:2, mol/mol) liposomes.
  • Figures 14A and 14B show a comparison of two T7 autogenes, pT7 AUTO 2C " and pCMV/T7-T7pol, for their ability to sustain expression of pT7-CAT or fT7-CAT over time ( Figure 14A) and their relative toxicities over time ( Figure 14B) .
  • Figures 15A, 15B, 16A and 16B show CAT activity
  • Figures 15A and 15B and protein recovered ( Figures 16A and 16B) in 2008, C3 , CHO and 293 cells transfected with pT7-CAT (1 ⁇ g/well) co-delivered to cells with increasing concentrations of pT7 AUTO 2C- ( Figures 15A and 16A) or pCMV/T7-T7pol ( Figures 15B and 16B) by DC-chol:DOPE liposomes at 10 nmol lipid/ ⁇ g DNA.
  • Figures 17A, 17B, 18A and 18B show CAT activity ( Figures 17A and 17B) and protein recovered ( Figures 18A and 18B) in 2008, C3, CHO and 293 cells transfected with pT7-CAT (1 ⁇ g/well) co-delivered to cells with increasing concentrations of pT7 AUTO 2C " ( Figures 17A and 18A) or pCMV/T7-T7pol ( Figures 17B and 18B) by LipofectAMINE at 5 nmol lipid/ ⁇ g DNA.
  • Figure 19 shows a comparison of the CAT activities of plasmid (pT7-CAT and pCMV-CAT) and linear
  • fT7-CAT and fCMV-CAT DNA sequences delivered by DC- chol:DOPE (3:2 mol/mol) liposomes or liposome/polylysine/ DNA ternary complex (LPD) .
  • 0.27 pmol of DNA (pT7-CAT, pCMV-CAT, fT7-CAt or fCMV-CAT) complexed with liposomal lipid (10 nmol lipid/ug DNA) or formulated in LPD complex was used to transfect 293-T7 cells (a 293 cell line stably transfected with pCMV/T7-T7pol) for 4 hours.
  • CAT assays were performed 48 hours following the transfection.
  • Figures 20A and 20B show CAT activity (Fig. 20A) in 293, BL6 and C3 cell lines.
  • fT7-CAT linear
  • pT7-CAT plasmid
  • Fig. 20B lipofectAMINE
  • FIGS 21A and 2IB pCMV/T7-T7pol produces higher levels of CAT activity than pT7 AUTO 2C-autoqene due to a higher production of T7 RNA polymerase.
  • 21A l ⁇ g of pT7-CAT was co-transfected with either 1) 150 U of T7 RNA polymerase alone; 2) increasing concentrations of pCMV/T7-T7pol or pT7 AUTO 2C alone; or 3) increasing concentrations of pCMV/T7-T7Pol or pT7 AUTO 2C " combined with 150 U of T7 RNA polymerase.
  • CAT assays were performed on cell lysates 2 days after transfection.
  • lane c pCMV/T7-T7pol
  • lane d pCMV/T7-T7pol + T7 RNA polymerase
  • lane e T7 AUTO 2C-
  • lane f T7 AUTO 2C- + T7 RNA polymerase.
  • FIGS 22A and 22B PCMV/T7-T7PO1 promotes high, sustained levels of T7 RNA polymerase and CAT expression.
  • 22A Time course analysis of CAT activity based on the same data shown in Figure 13A. The data of Figure 22A is presented as total CAT activity rather than per mg protein as shown in Figure 13A. l ⁇ g of pT7-CAT was combined with either 0.3 ⁇ g of pCMV/T7-T7pol or 0.3 ⁇ g of pT7 AUTO 2C " + 150 U of T7 RNA polymerase and transfected to 293 cells via DC-chol:DOPE liposomes (l ⁇ g DNA/lOnmol lipid) .
  • FIG. 23 pT7-CAT/pCMV/T7-T7pol (cytoplasmic expression) induces higher levels of CAT expression when compared with pCMV-CAT (nuclear expression) .
  • l ⁇ g of pT7- CAT was combined with 0.3, 0.7, 1.0 or 2.0 ⁇ g of pCMV/T7- T7pol and transfected to 293 cells by DC-chol:DOPE liposomes (l ⁇ g DNA/lOnmol lipid) for 4 h at 37°C.
  • DC-chol:DOPE liposomes l ⁇ g DNA/lOnmol lipid
  • CAT activities were determined 2 days after transfection.
  • Figure 24 Time Course Comparing PT7- CAT/pCMV/T7-T7pol fcvtoplasmic expression) and pCMV-CAT (nuclear expression) in 293 cells.
  • l ⁇ g of pT7-CAT was transfected to 293 cells with either 0.7, 1.0 or 2.O ⁇ g of pCMV/T7-T7pol via DC-chol:DOPE liposomes (l ⁇ g DNA/lOnmol lipid) for 4 h at 37°C.
  • l ⁇ g of pCMV-CAT was complexed to DC-chol:DOPE liposomes and delivered to 293 cells in the same manner. Cells were lysed daily up to 7 days after transfection and were split 1:1 at day 4 after transfection.
  • Figures 25A and 25B DNA/Protamine/Liposome Complexes Can Enhance Can Enhance Expression in the Cytoplasmic Expression System.
  • l ⁇ g of pT7-CAT (25A) or pCMV-CAT (25B) was combined with 0 or 2 ⁇ g of protamine before complexing with one of the following liposome formulations (l ⁇ g DNA/lOnmol lipid) : DOTAP (2 ⁇ mol/ml) , DOTAP:DOPE (2 ⁇ mol/ml) , LipofectAMINE (2.34 ⁇ mol/ml) , DC- chol:DOPE (2 ⁇ mol/ml) , and Lipofectin (1.6 ⁇ mol/ml) .
  • DOTAP (2 ⁇ mol/ml)
  • DOTAP:DOPE 2 ⁇ mol/ml
  • LipofectAMINE 2.34 ⁇ mol/ml
  • DC- chol:DOPE 2 ⁇ mol/ml
  • Lipofectin 1.6 ⁇ mol/ml
  • FIG. 26 Sequencing results for Hindlll junction site in pCMV/T7-T7pol.
  • the T7 gene 1 from pAR3126 was cloned as a Hindlll/BamHl fragment into a pcDNA vector backbone.
  • the sequence between the J brackets is the junction site.
  • the sequence between the A brackets is the T7 promoter from pcDNA3 and sequence between the B brackets is the Hindlll site.
  • Figure 27 Sequencing results for BamHl junction site in pCMV/T7-T7pol.
  • the sequence between the J brackets is the junction site of the T7 gene 1 from pAR3126 cloned as a Hindlll/BamHI fragment into the pcDNA3 vector.
  • the sequence between the C brackets is the BamHl site.
  • Figure 28 Hindlll/BamHl fragment sequence from pAR3126 containing the T7 gene 1.
  • the present invention relates to a cytoplasmic gene expression system for use in eukaryotic cells.
  • Gene transfer with cytoplasmic expression systems can be more advantageous than with nuclear expression systems for a number of reasons.
  • many non-viral gene transfer vectors have been developed to improve DNA delivery to the cytoplasm of cells; however, there have been no significant advances in improving the nuclear transport of DNA. It has been previously shown that nuclear transport of cytoplasmic DNA is of very low efficiency (Capecchi (1980) Cell 22, 479-488). Therefore, the overall expression of DNA is hindered by inefficient transport of DNA into the nucleus where the transcription machinery resides.
  • the cytoplasmic expression systems of the invention rely on a dual promoter prokaryotic expression 5 in the cytoplasm of cells without the addition of exogenous prokaryotic RNA polymerase.
  • This autogene contains a eukaryotic promoter which drives the initial expression of prokaryotic RNA polymerase in the nucleus. Though nuclear delivery of the autogene is thought to be
  • prokaryotic RNA polymerase 10 poor, enough prokaryotic RNA polymerase can be produced in the cytoplasm via a second prokaryotic promoter within the autogene. Therefore, the expression of prokaryotic RNA polymerase from this autogene is mostly driven in the cytoplasm by the prokaryotic promoter.
  • polymerase generated from the autogene is then able to induce sustained expression of a co-transfected nucleic acid sequence operably linked to the same prokaryotic promoter.
  • Such autogenes are therefore useful in, for example, gene therapy/transfer applications in which high
  • target sequence as used throughout the specification and claims is meant a double-stranded DNA sequence whose expression is desired.
  • suitable target sequences for use in the invention are
  • the autogene of the invention comprises, at a minimum, the following elements operatively linked: a nucleic acid sequence encoding a prokaryotic RNA polymerase gene, a cognate promoter of the RNA polymerase and a promoter capable of functioning in eukaryotic cells, e.g., a eukaryotic promoter.
  • operatively linked is meant that the elements are in a spatial arrangement which results in expression of the RNA polymerase at levels sufficient to initiate transcription, through the cognate promoter, of the nucleic acid sequence encoding the RNA polymerase.
  • the autogenes of the invention may be in circular form as plasmid DNA or in linear form such as chemically synthesized DNA or PCR amplification products.
  • the autogenes of the invention may be used to sustain the expression of the target sequence present in plasmid DNA.
  • RNA polymerase encoded by the invention is preferably a single polypeptide enzyme that is capable of recognizing its cognate promoter sequence with high affinity and specificity and that does not require host cell factors to initiate transcription from the cognate promoter.
  • RNA polymerases suitable for use in the autogenes of the invention include, but are not limited to, the bacteriophage RNA polymerases T7, SP6 and T3 and mitochondrial RNA polymerases.
  • the "eukaryotic promoter" of the autogenes of the invention may be any promoter which is capable of initiating transcription in the nucleus of eukaryotic cells.
  • cognate promoter any promoter sequence which is recognized by the RNA polymerase encoded by the autogene.
  • the cognate promoter sequence may be a promoter sequence cloned from the genome of the RNA polymerase or it may be modified by mutations in order to reduce the level of expression of RNA polymerase from the autogene and hence, reduce toxicity of the autogenes to E. coli and mammalian cells.
  • the autogene of the invention comprises in 5' to 3' order: the following elements operatively linked: a eukaryotic promoter, a cognate promoter for the RNA polymerase, and a nucleic acid sequence encoding the RNA polymerase.
  • the RNA polymerase is bacteriophage T7 RNA polymerase
  • the cognate promoter is a T7 promoter
  • CMV cytomegalovirus
  • An example of a preferred RNA polymerase autogene is the pCMV/T7-T7 pol autogene shown in Figure 2. This autogene has been demonstrated to be nontoxic to both E.
  • RNA polymerase from the autogenes of the invention may be modulated by modification of the minimal elements present in the autogene (i.e. the eukaryotic promoter, the cognate promoter and the RNA polymerase gene) or by the insertion of additional elements into the autogene.
  • mutant RNA polymerases can be designed which are capable of binding to their cognate promoter and carrying on transcription without being lethal to a bacterial host (U.S.
  • Patent 5,122,457 and the wild-type cognate promoters may be mutated (preferably by point mutations) to reduce transcription of the gene operably linked to the cognate promoter.
  • mutations of the T7 promoter include, but are not limited to, m2T7 p and M3T7p which have been shown to have 1% and 10% the activity of wild-type T7 promoter in in vitro transcription assays.
  • the cognate promoter may be modified by attaching at its 3' end a bacterial operator sequence such as the lac operator where the operator may serve to reduce the toxicity of autogene constructs in the bacterial host.
  • an autogene of the invention may comprise the following elements operatively linked: a promoter active in eukaryotic cells (e.g., a eukaryotic promoter) a modified cognate promoter and a nucleic acid sequence encoding the RNA polymerase.
  • the modified or unmodified cognate promoter may be linked at its 3' end to the 5' end of an internal ribosome entry site sequence (IRES) in order to enhance translation of the RNA polymerase gene by enabling the formation of translational initiation complex in eukaryotic cells without the need for the RNA to be capped at its '5 end.
  • the IRES is a priornaviral IRES, most preferably an encephalomyocarditis (EMC) IRES sequence.
  • the RNA polymerase gene may be linked at its 3' end to its cognate transcription terminator sequence in order to enhance the fidelity of transcription of the RNA polymerase gene.
  • terminator sequences can be cloned from the genome encoding the RNA polymerase by, for example, physically mapping the 3 ' end of RNA transcripts by methods known to those of ordinary skill in the art or, where the sequence of the cognate terminator is known, the sequence may be synthesized.
  • autogene constructs in which the cognate promoter is linked to an IRES and the 3' end of the polymerase gene is linked to a terminator sequence are shown in Figure 3 where the autogene construct comprises in 5' to 3 ' order: a CMV immediate early promoter, a mutant T7 promoter, an EMC IRES, sequence the T7 RNA polymerase gene and a T7 terminator and in Figure 4, where the autogene comprises in 5' to 3' order: a CMV promoter, a T7 promoter fused to the lac operator, an EMC IRES sequence, a T7 RNA polymerase gene, and a T7 terminator.
  • an autogene construct of the invention may be designed to contain either an IRES or a terminator sequence or both.
  • the present invention also relates to cytoplasmic expression systems which utilize the RNA polymerase autogenes of the invention.
  • the cytoplasmic expression system is a dual sequence cytoplasmic expression system which consists of a first nucleic acid sequence comprising a eukaryotic promoter operatively linked to a cognate promoter which is operatively linked to a prokaryotic RNA polymerase gene and a second sequence which comprises a second copy of the cognate promoter of the first sequence operatively linked to the target sequence.
  • the present invention also provides a "single sequence cytoplasmic expression system" in which the first and second sequences described above are contained in a single nucleic acid molecule.
  • a single sequence expression system is provided in Figures 5 and 6.
  • the first copy of the cognate promoter may be a mutant promoter and the second copy of the cognate promoter (driving expression of the target sequence) may be a wild-type promoter.
  • the single sequence system may be more efficient for introduction into host cells than the dual system composed of two separate plasmids.
  • the dual system may be of particular utility in situations where it is desirable to adjust the ratio of expressed RNA polymerase and the target sequence of interest by providing different ratios of the two plasmids.
  • the present invention also relates to the use of the self-initiating and self-sustaining autogene in the above single sequence or double sequence expression systems to express other gene products including proteins, anti-sense molecules or ribozymes in eukaryotic cells.
  • Such expression systems encompass a wide variety of applications. For example, it is especially suitable for expressing genes in a specific cell type where appropriate promoters for that cell type are unavailable.
  • the autogenes may therefore be used for synthesizing desired proteins in vitro or in vivo. It is also useful in vitro for the transient expression of a newly cloned gene for confirmation of the identity of its encoded gene product as well as the rapid production of high amounts of proteins for use in immunization of animals for the generation of specific antibodies.
  • the present invention may be used to introduce exogenous genes into cells and tissues in vivo for transient expression of the gene product to elicit an antigen-specific host immune response.
  • the short-term nature of gene expression may be ideal for its use as a vehicle for jjn vivo immunization of a host to an antigen.
  • a major impediment in the current attempts of gene therapy is the integration of foreign genes in non- dividing eukaryotic host cells.
  • the ability of the present system to permit gene expression in the cell cytoplasm circumvents this potential problem, as gene expression can be achieved in quiescent cells.
  • the present invention is also self-limiting.
  • the self-limiting nature of this expression system is particularly suitable for use in settings where gene expression is desirable only temporarily or transiently.
  • the present invention may be used to target lymphokines genes to tumor sites in vivo for the in situ activation of cytotoxic lymphocytes to mediate tumor cytolysis.
  • the single or dual sequence expression systems may be suspended in saline, PBS, serum free media or any aqueous solution suitable for in vivo administration.
  • the solution may be injected into an animal, including a human, intravenously, intramuscularly, intracranially, 0 subcutaneously or directly into a tissue or organ.
  • the single or dual sequence systems may be injected alone, encapsulated by liposomes or linked to any carrier molecules capable of translocating the system across the plasma membrane.
  • the 5 expression system may be injected in the dose range of
  • the systems may be injected intravenously to cause endothelial cell uptake and release of the expression o product directly into the circulation.
  • PCR polymerase chain reaction
  • CAT E coli chloramphenicol acetyltransferase
  • CMV cytomegalovirus immediate early promoter
  • T7 bacteriophage T7 promoter
  • T7pol bacteriophage T7 RNA polymerase coding gene
  • DC- chol 30[N-(N' ,N'-dimethylaminoethane)-carbamoyl] cholesterol
  • DOPE l,2-dioleoyl-sn-glycero-3-phosphatidyl- ethanolamine
  • LPD liposome/polycation/DNA ternary complex
  • CHEMS cholesteryl hemisuccinate
  • T7 RNA polymerase (New England Biolabs, Beverly, MA) was used without further purification. All the chemicals for PCR including Taq polymerase, nucleotides and buffer were purchased from Gibco BRL (Gaithersburg, MD) . Acetyl coenzyme A, chloramphenicol, Triton X-100 and the hydrobromide salt of the polycation poly-L-lysine (MW 25,600) were from Sigma (St Louis, MO). [ 3 H]acetyl coenzyme A and Beta-Max scintillation cocktail were from ICN Biomedicals (Costa Mesa, CA) .
  • Dioleoylphosphatidylethanolamine (DOPE) and cholesteryl-hemisuccinate (CHEMS) were obtained from Avanti Polar Lipids, Inc (Birmingham, AL) .
  • DC-chol 3/3[N-(N' ,N'- dimethylaminoethane)-carbamoyl] cholesterol (DC-chol) was synthesized according to Gao and Huang ((1991) Biochem. Biophvs. Res. Commun. 179: 280-285).
  • Unilamellar DC- chol:DOPE liposomes of approximately 150 nm in diameter were prepared by microfluidization of a hydrated mixture of DC-chol and DOPE (3:2, m/m) , and filter sterilized.
  • pH-sensitive liposomes (approximately 120 nm in diameter) composed of CHEMS and DOPE (3:2, mol/mol) were prepared by sonication.
  • LipofectAMINE was obtained from Gibro BRL.
  • PCR For PCR, 10 ng of plasmid template, 50 pmoles of each primer (shown in Figure 1), 0.2 mM dNTPs, and 2.5 units of Thermus aquaticus (Ta ⁇ ) DNA polymerase were used.
  • the PCR protocol consists of 4 min of denaturation at 94°C, then 30 cycles of 45 sec at 94°C, 1 min at 68°C, and 2 min at 72°C and finally extension of 5 min at 72°C.
  • the resulting PCR amplified fragments were extracted with phenol-chloroform, ethanol-precipitated, resuspended in water and then further purified by filtration through a ° size exclusion filter to remove unused primers and free oligonucleotide.
  • Plasmid pT7-CAT which is the same as pT7-EMC- CAT, a plasmid containing the CAT reporter gene driven by the bacteriophage T7 promoter (Elroy-Stein, et al. (1989) 5 Proc. Natl. Acad. Sci. USA 86: 6126-6130) , was a kind gift provided by Dr. B. Moss (National Institutes of Health) .
  • pT7 AUTO 2C " an autogene composed of the T7 RNA polymerase gene and its corresponding promoter, was maintained and purified as previously described by Gao et 0 al (Gao, X., et al. (1994) Biochem. Biophys. Res. Commun.
  • Plasmid pUCCMV-CAT was constructed by Dr. H. Farhood (Farhood, H., et al. (1995) Biochem. Biophvs. Acta.. 1235: 289-295) .
  • the pCMV/T7-T7pol autogene was constructed by inserting a Hindlll/BamHI 5 fragment from pAR3126 (Dunn, J.J., et al. (1988) Gene 68: 259-266) containing the cDNA of T7 RNA polymerase into the corresponding sites of the pcDNA3 plasmid vector (Invitrogen Corp., San Diego, CA) (See Figures 2, 26-28) .
  • This autogene was amplified in E. coli DH5 ⁇ F' cells and o purified by CsCl gradient centrifugation method (Sambrook, J., et al. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press: Plainview, NY) .
  • LPD complex The molecular weight of the hydrobromide salt of poly-L-lysine used to produce the LPD was 25,600. LPD complex was prepared prior to transfection by mixing pH- sensitive liposomes containing CHEMS and DOPE and the cationic DNA/polylysine (1:0.75, w/w) complex. The lipid to DNA ratio in the final LPD complex was 3 : 1 (w/w) . The resulting LPD complex had a cationic charge.
  • 293-T7 a 293 cell line which produces endogenous T7 RNA polymerase, was established by co-transfecting 293 cells with pCMV/T7-T7pol and pBK-CMV (Neo r ) phagemid followed by selection by G418.
  • the 293-T7 cells were then cultured in DMEM medium (will serum and antibiotics as for 293 cells) supplemented with G418 (0.4 mg/ml).
  • RNA polymerase was co-delivered with T7-EMC-CAT (plasmid or PCR fragment) .
  • the medium serum-free media
  • normal growth medium containing 10% fetal bovine serum and antibiotics
  • cells were cultured for 2 days before the CAT assay was performed.
  • cells were collected daily for 9 days, and were split 1:1 once on day 4 when they were 100% confluent.
  • 293 cells were grown to 70-80% confluency in 24 well plates.
  • pT7-CAT (l ⁇ g) diluted in serum free DMEM medium was mixed with either T7 RNA polymerase (150 U, New England Biolabs, Beverly, MA) , 0.3 ⁇ g of either autogene (pCMV/T7-T7pol or pT7 AUTO 2C " ) or a combination of enzyme and autogene before complexing with DC-chol:DOPE liposomes (10 nmol total lipid) .
  • This mixture was used to transfect cells for 4 hours at 37°C after which transfection medium (serum-free medium) was replaced with growth medium (DMEM containing 10% FBS plus antibiotics) .
  • transfection efficiency studies cells were lysed for CAT assays 48 hours following transfection. For time course studies, cells were split once at day 4 and were collected daily up to 7 days after transfection for CAT assay.
  • C3, 293, and CHO cell lines were cultured to -70% confluency in 24 well plates prior to transfection.
  • pT7-CAT 15 pT7-CAT (l ⁇ g/well) was co-delivered to cells with increasing concentrations of pCMV/T7-T7pol or pT7 AUT02C- autogene T7 RNA polymerase (150U) by either DC- chol liposomes (ionmol lipid/mgDNA) or LipofectAmine (5 nmol lipid/ ⁇ g DNA) . After 4 hr, transfection medium was
  • pT7-CAT or fT7-CAT (0.27 pmol/well) was co-delivered to cells with pCMV/T7-T7pol autogene (0.3 ⁇ g/well) via DC-chol liposomes or LipofectAMINE (10 nmol lipid/ ⁇ g DNA) .
  • pCMV/T7-T7pol autogene 0.3 ⁇ g/well
  • LipofectAMINE 10 nmol lipid/ ⁇ g DNA
  • the reaction was performed at 37°C for 1 hour and the product of the reaction, acetylated chloramphenicol (1,3-diacetylchloramphenicol) , was then extracted from the aqueous phase with 1 ml of toluene. One half of the organic phase was then mixed with 3 ml of Beta-MaxTM (ICN Biomedicals, Costa Mesa, CA) and counted for radioactivity. One unit was defined as the amount of enzyme converting l nmol of acetyl groups to chloramphenicol per minute under the above reaction conditions.
  • T7-CAT DNAs Cytoplasmic expression of T7-CAT DNAs was investigated by examining expression of the CAT reporter gene in 293-T7 cells as a function of pT7-CAT or fT7-CAT DNA concentrations. As shown in Figure 7, the CAT activity increased with increasing amounts " of either pT7-CAT or fT7-CAT, but reached a plateau when the T7-CAT DNA concentrations were above 0.54 pmol. No significant difference was found between CAT activity for pT7-CAT and fT7-CAT when delivered by DC-chol:DOPE liposomes.
  • CAT expression from pT7- CAT or fT7-CAT peaked at day 5 and declined slowly thereafter to a level of expression at day 9 that was about 1/4 of the peak level.
  • This duration of CAT expression is longer than that seen with the nuclear expression system which is usually 3-5 days (Gao, X. , et al. (1994) Biochem. Biophys. Res. Commun. , 200: 1201- 1206) ; a result that might be due to the fact that some of the T7-CAT remained associated with liposomes following release into the cytoplasm thereby preventing the DNA from being rapidly degraded by cytoplasmic enzymes.
  • CAT Reporter Gene could be expressed in 293 cells which do not stably express T7 RNA polymerase
  • pT7-CAT or fT7-CAT were co- delivered into 293 cells with either purified T7 RNA polymerase or the T7 autogene pCMV/T7-T7pol.
  • Table 1 clearly show that CAT expression for both plasmid and linear DNA fragments were dramatically lower when the source of T7 RNA polymerase was the exogenously supplied pure protein as compared to the T7 autogene.
  • T7-CAT (1 ⁇ g) was co-transfected with either pure T7 RNA polymerase (150U/well) or pCMV/T7-T7pol (0.3 ⁇ g/well) to 293 cells with DC-chol:DOPE liposomes (10 nmol) for 4 hours. CAT activity was assayed 48 h after transfection.
  • the pCMV/T7-T7pol autogene was employed as a source of T7 RNA polymerase in subsequent studies.
  • CAT activity increased in normal 293 cells co-transfected with varying amounts of pT7-CAT or fT7-CAT and pCMV/T7-T7pol (0.3mg) in a fashion slightly different from that observed earlier in 293-T7 cells. This result might be due to the difference in the amount of intracellular T7 RNA polymerase expressed in 293 and T7-293 cells. No toxicity was found at any dose tested.
  • pCMV/T7-T7pol produced a higher level of CAT gene expression as compared with that seen with the pT7AUT02C " autogene.
  • This elevated CAT gene expression with the pCMV/T7-T7pol autogene did not require any additional T7 RNA polymerase to drive the T7 RNA polymerase regeneration process.
  • additional enzyme was added, there was a further increase in CAT activity, suggesting that exogenous T7 RNA polymerase delivered to the cytoplasm is acting on the T7 promoter to promote autogene expression and thus induce higher reporter gene expression than that seen with pCMV/T7-T7pol alone.
  • CAT activity with pT7AUT02C ⁇ was only seen in the presence of additional enzyme and the CAT activity was lower than that observed with the pCMV/T7-T7pol autogene. Both autogenes, however, demonstrated much higher CAT activity than that seen with T7 RNA polymerase alone. This result suggests that a constant production of T7 RNA polymerase is required for higher levels of gene expression in the cytoplasm.
  • Example 9 pCMV/T7-T7pol autogene is less toxic and more efficient in its expression of PT7-CAT than pT7AUT02C in 4 different cell lines
  • T7 AUTO 2C" autogenes co-delivered with pT7-CAT was compared in 2008, C3, CHO, and 293 cells in Figures 15-18. It is obvious that the pCMV/T7-T7pol autogene induced considerably higher levels of CAT expression in all the cell lines used as compared to pT7AUT02C " (compare Figures 15B and 17B with Figures 15A and 17A) . This expression seems to be formulation dependent since LipofectAMINE seemed to induce even higher levels of CAT activity than that seen with DC-chol liposomes for both autogenes (compare Figures 14A and 14B with Figures 15A and 15B) .
  • LipofectAMINE and its ability to further condense DNA for cellular entry.
  • T7-CAT plasmid DNA
  • fT7-CAT linear PCR-generated DNA fragments
  • CMV-CAT nuclear expression systems
  • Example 9 To determine if the low CAT expression obtained 10 in Example 9 for fCMV-CAT relative to that observed for pCMV-CAT might have resulted from the lack of an optimized delivery system for linear DNA transport to the nucleus, the following experiment was conducted: pCMV-CAT or fCMV-CAT (nuclear expression system) or pT7-CAT or fT7-CAT l ⁇ (cytoplasmic expression system) codelivered with pCMV/T7- T7pol autogene were delivered to 293, BL6 or C3 cells by either DC-chol liposomes or lipofectAMINE (10 nmol lipid/1 ⁇ g DNA) , and CAT activity was as ⁇ ayed 48 hours after transfection.
  • pCMV-CAT or fCMV-CAT nuclear expression system
  • pT7-CAT or fT7-CAT l ⁇ cytoplasmic expression system
  • Mice are injected intravenously, intramuscularly, intracranially, subcutaneously or directly into the tissue or organ with complexes of DC- chol liposomes and transgenes driven by the T7 promoter (plasmid or linear) alone or combined with pCMV/T7-T7pol autogene (plasmid or linear) . About 100 ⁇ g total DNA is injected. Animals are sacrificed 2 days later and extracts of tissues of interest (liver, kidney, lung, heart, brain, etc.) are assayed for CAT activity. CAT activity is determined in various tissues.
  • pCMV/T7-T7pol can produce high levels of endogenous T7 RNA polymerase in the presence or absence of exogenous T7 RNA polymerase; however, endogenous T7 RNA polymerase transcribed from pT7 AUTO 2C" was too low to be detected. Therefore, the pCMV/T7-T7pol autogene can induce high CAT activity in cells transfected with pT7-CAT due to the high production of T7 RNA polymerase transcribed by pCMV/T7-T7pol.
  • Example 15 The PCMV-T7-T7 pol Cytoplasmic Expression System Can Produce Higher Levels of Activity Than Other Cytoplasmic or Nuclear Expression Systems
  • the cytoplasmic expression system pT7-CAT + pCMV/T7-T7pol was compared with a nuclear expression vector pCMV-CAT to determine if the cytoplasmic system can increase transcription/translation of the reporter gene CAT over that seen with the strong nuclear cytomegalovirus promoter.
  • the addition of pCMV/T7-T7pol at concentrations above 0.3 ⁇ g produced higher CAT activity than pCMV/CAT.

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Abstract

L'invention concerne de nouveaux autogènes procaryotes d'ARN polymérase qu'on peut utiliser dans des systèmes eucaryotes d'expression cytoplasmique.
PCT/US1997/007030 1996-04-26 1997-04-25 Systeme d'expression cytoplasmique de gene mettant en application un autogene procaryote d'arn polymerase WO1997041243A2 (fr)

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AU27446/97A AU2744697A (en) 1996-04-26 1997-04-25 A cytoplasmic gene expression system which utilizes a prokaryotic rna polymerase autogene
JP09539094A JP2000510334A (ja) 1996-04-26 1997-04-25 原核生物ｒｎａポリメラーゼ自己遺伝子を利用する細胞質遺伝子発現系
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WO1999057290A1 (fr) 1998-05-05 1999-11-11 Hsf Pharmaceuticals Sa Circuits moleculaires regulateurs permettant d'obtenir une activation soutenue de genes d'interet par un seul stress
WO2002088370A2 (fr) * 2001-04-30 2002-11-07 Protiva Biotherapeutics Inc. Acides nucleiques autogenes codants pour une polymerase d'arn secretable
WO2005035764A1 (fr) * 2003-10-16 2005-04-21 The University Of British Columbia Acides nucleiques autogenes codant un arn polymerase pouvant etre secrete
US6916653B2 (en) 1998-01-15 2005-07-12 King's College London Ribozymal nucleic acid
WO2005090568A2 (fr) * 2004-03-20 2005-09-29 University Of Sheffield Reaction en chaine de promoteur
WO2016174195A1 (fr) * 2015-04-30 2016-11-03 Universität Für Bodenkultur Wien Croissance de découplage et production de protéines

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6916653B2 (en) 1998-01-15 2005-07-12 King's College London Ribozymal nucleic acid
WO1999057290A1 (fr) 1998-05-05 1999-11-11 Hsf Pharmaceuticals Sa Circuits moleculaires regulateurs permettant d'obtenir une activation soutenue de genes d'interet par un seul stress
EP1075529B1 (fr) * 1998-05-05 2013-01-16 Richard Voellmy Circuits moleculaires regulateurs permettant d'obtenir une activation soutenue de genes d'interet par un seul stress
WO2002088370A2 (fr) * 2001-04-30 2002-11-07 Protiva Biotherapeutics Inc. Acides nucleiques autogenes codants pour une polymerase d'arn secretable
WO2002088370A3 (fr) * 2001-04-30 2003-09-25 Protiva Biotherapeutics Inc Acides nucleiques autogenes codants pour une polymerase d'arn secretable
US7364750B2 (en) 2001-04-30 2008-04-29 The University Of British Columbia Autogene nucleic acids encoding a secretable RNA polymerase
WO2005035764A1 (fr) * 2003-10-16 2005-04-21 The University Of British Columbia Acides nucleiques autogenes codant un arn polymerase pouvant etre secrete
WO2005090568A2 (fr) * 2004-03-20 2005-09-29 University Of Sheffield Reaction en chaine de promoteur
WO2005090568A3 (fr) * 2004-03-20 2005-11-03 Univ Sheffield Reaction en chaine de promoteur
WO2016174195A1 (fr) * 2015-04-30 2016-11-03 Universität Für Bodenkultur Wien Croissance de découplage et production de protéines
US11046963B2 (en) 2015-04-30 2021-06-29 Engenes Biotech Gmbh Uncoupling growth and protein production

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