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WO1997041243A9 - 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

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Publication number
WO1997041243A9
WO1997041243A9 PCT/US1997/007030 US9707030W WO9741243A9 WO 1997041243 A9 WO1997041243 A9 WO 1997041243A9 US 9707030 W US9707030 W US 9707030W WO 9741243 A9 WO9741243 A9 WO 9741243A9
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WO
WIPO (PCT)
Prior art keywords
sequence
cat
cells
promoter
rna polymerase
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US1997/007030
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English (en)
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WO1997041243A3 (fr
WO1997041243A2 (fr
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Publication date
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Priority to EP97921403A priority Critical patent/EP0910653A2/fr
Priority to AU27446/97A priority patent/AU2744697A/en
Priority to JP09539094A priority patent/JP2000510334A/ja
Publication of WO1997041243A2 publication Critical patent/WO1997041243A2/fr
Publication of WO1997041243A9 publication Critical patent/WO1997041243A9/fr
Publication of WO1997041243A3 publication Critical patent/WO1997041243A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Definitions

  • 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.
  • 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.
  • 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.
  • 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 0 promoter is separated from the target sequence in the dual and single sequence systems by an IRES.
  • 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 PAUT0M2-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 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- chol:D0PE 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 11A and 11B show CAT expression (Figure 11A) 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 11A CAT activity was determined 48 hours after transfection and in Figure 11B, 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 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 2OB 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-autocrene 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.
  • 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.
  • 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 (cytoplasmic 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.
  • 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.
  • Figure 27 Seguencing results for BamHI junction site in pCMV/T7-T7pol.
  • the sequence between the J brackets is the junction site of the T7 gene l from pAR3126 cloned as a Hindlll/BamHI fragment into the pcDNA3 vector.
  • the sequence between the C brackets is the BamHI site.
  • 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 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.
  • 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.
  • 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 5 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
  • 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 in vivo immunization of
  • a major impediment in the current attempts of gene therapy is the integration of foreign genes in non- dividing eukaryotic host cells.
  • 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) .
  • 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.
  • C3, 293, and CHO cell lines were cultured to ⁇ 70% confluency in 24 well plates prior to transfection.
  • T7-CAT plasmid DNA
  • fT7-CAT linear PCR-generated DNA fragments
  • CMV-CAT nuclear expression systems

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 Ceased WO1997041243A2 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP97921403A EP0910653A2 (fr) 1996-04-26 1997-04-25 Systeme d'expression cytoplasmique de gene mettant en application un autogene procaryote d'arn polymerase
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

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US1630496P 1996-04-26 1996-04-26
US60/016,304 1996-04-26

Publications (3)

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WO1997041243A2 WO1997041243A2 (fr) 1997-11-06
WO1997041243A9 true WO1997041243A9 (fr) 1998-01-22
WO1997041243A3 WO1997041243A3 (fr) 1998-02-19

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EP (1) EP0910653A2 (fr)
JP (1) JP2000510334A (fr)
AU (1) AU2744697A (fr)
CA (1) CA2250759A1 (fr)
WO (1) WO1997041243A2 (fr)

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JP4312957B2 (ja) 1998-01-15 2009-08-12 キングス・カレツジ・ロンドン Ｃｃｒ５あるいはｃｘｃｒ４を開裂するリボザイム核酸
US6342596B1 (en) * 1998-05-05 2002-01-29 Hsf Pharmaceuticals S.A. Molecular regulatory circuits to achieve sustained activation of genes of interest by a single stress
US7364750B2 (en) 2001-04-30 2008-04-29 The University Of British Columbia Autogene nucleic acids encoding a secretable RNA polymerase
US20040142892A1 (en) * 2001-04-30 2004-07-22 The University Of British Columbia Autogene nucleic acids encoding a secretable RNA polymerase
GB0406330D0 (en) * 2004-03-20 2004-04-21 Univ Sheffield Gene screen
DK3289088T3 (da) 2015-04-30 2020-06-29 Engenes Biotech Gmbh Afkobling af vækst fra proteinproduktion

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DE69022165T2 (de) * 1989-10-31 1996-04-04 Sagami Chem Res Klonierungs-Plasmid-Vektor, Primer-Vektor davon und Herstellungsverfahren einer cDNA-Bank.
CA2085353A1 (fr) * 1990-06-18 1991-12-19 Chandrakant P. Giri Systeme de vecteurs d'expression chez les eucaryotes
US5591601A (en) * 1993-05-14 1997-01-07 Ohio University Edison Animal Biotechnology Institute DNA polymerase gene expression system utilizing an RNA polymerase co-delivered with the gene expression vector system
CA2140081C (fr) * 1994-01-13 2008-04-01 Dean L. Engelhardt Procede, construct et conjugue permettant de produire des copies multiples d'acides nucleiques

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