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WO2002028168A1 - Systeme d"expression de genes regulables a haut rendement - Google Patents

Systeme d"expression de genes regulables a haut rendement Download PDF

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Publication number
WO2002028168A1
WO2002028168A1 PCT/US2001/031138 US0131138W WO0228168A1 WO 2002028168 A1 WO2002028168 A1 WO 2002028168A1 US 0131138 W US0131138 W US 0131138W WO 0228168 A1 WO0228168 A1 WO 0228168A1
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nucleic acid
promoter
teto
transactivator
tetracycline
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PCT/US2001/031138
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English (en)
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Aha-Mohammadi Siamak
Michael T. Lotze
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University Of Pittsburgh Of The Commonwealth System Of Higher Education
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Priority to US10/148,521 priority Critical patent/US20030221203A1/en
Priority to AU2001296607A priority patent/AU2001296607A1/en
Publication of WO2002028168A1 publication Critical patent/WO2002028168A1/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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
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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
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    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus
    • 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
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/027—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a retrovirus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/15—Vector systems having a special element relevant for transcription chimeric enhancer/promoter combination

Definitions

  • a high efficiency regulatable gene expression system including a promoter and a humanized transactivator. Also provided are methods for inducing expression of a nucleic acid using the regulatable gene expression system.
  • TRS tetracycline-regulatable systems
  • Vaccination with irradiated tumor cells engineered to secrete murine granulocyte-macrophage colony-stimulating factor stimulates potent, specific, and long lasting anti-tumor immunity. Proc. Natl . Acad. Sci . USA.
  • chimeric regulatable systems incorporating various prokaryotic and eukaryotic elements have been devised to overcome the poor efficiency and generalized pleiotropic effects of earlier regulatable promoters.
  • These systems employ drugs or hormones (or their analogs) as their inducing agents and utilize modular transcriptional transactivators composed of natural or mutant drug/hormone ligand binding domains, intrinsic or extrinsic DNA binding domains, and transcriptional activation domains and include, without limitation, the tetracycline-regulatable system, the progesterone-regulatable system, the ecdysone-regulatable system and the rapamycin-regulatable system (see, Agha- Moham adi, S., and Lotze, M.T.
  • Tetracycline- regulatable Vectors and systems as described above are commercially available from BD Biosciences Clontech ("Clontech”) under the trade names TET-OFFTM (tetracycline turns off gene expression) and TET-ONTM (tetracycline turns on gene expression) .
  • tetracycline-responsive promoters in a variety of applications, including: cell and gene therapies; veterinary uses; yeast expression systems; gene function studies; conditional immortalization of primary cells of therapeutic relevance; biocatalysis, fermentation and bioprocessing of therapeutic or other molecules; transgenic plants and animals; high throughput screening applications; functional genomics and target validation; and in plant biosciences, including establishment of disease and pest resistance.
  • TRS The broadly available first-generation tetracycline-regulatable gene expression system
  • TRS uses a conditionally active chimeric tTA (tetracycline transactivator) which was created by fusing the C-terminal 127 amino acids of herpes simplex virus VP16 to the carboxyl terminus of the tetR.
  • tetR tetracycline transactivator
  • the tetR moiety of tTA binds with high affinity and specificity to a regulatory region comprising 7 repeats of a B2 tetO sequence built upstream of a minimal human CMV promoter to form tetracycline-regulated promoter (tRP) .
  • the palindromic centers of each adjacent pair of tetO sequences is 41 bases apart, or about four turns of the DNA helix.
  • the VP16 moiety of tTA strongly induces transactivation of the distal target gene by promoting the assembly of a transcriptional initiation complex. Binding of tetracycline to tTA leads to a conformational change in tetR accompanied with loss of tetR affinity for tetOs (Gossen and Bujard, 1992) .
  • the TRS offers several advantages that make it particularly suitable for mammalian gene regulation (Agha- Mohammadi and Lotze, 2000) .
  • TRS the most important feature of TRS is its reliance on the tetracycline family of antibiotics as inducing agents .
  • the well documented pharmacokinetics and pharmacodynamics of these drugs enable their use in an optimized fashion in human subjects. Furthermore, these agents cause little adverse effects on mammalian cells at the concentrations utilized for TRS modulation. Also, the system displays unique kinetics of regulation in response to different clinically used tetracycline analogs .
  • the system uses oxytetracycline, displays rapid switch-off and switch-on kinetics within 72 hours (Agha- Mohammadi, S., Alvarez-Vallina, L., Ashworth, L.J., and Hawkins, R.E. (1997). Delay in resumption of the activity of tetracycline-regulatable promoter following removal of tetracycline analogues. Gene Ther. 4 ,993-997 , incorporated herein by reference in its entirety) . Apart from these benefits, the system can be delivered conveniently as a single vector (Agha-Mohammadi, S., and Hawkins, R.E. (1998).
  • pSialV The maximal transcriptional activity of pSialV was comparable to that of CMV IE promoter and its basal activity as low as the leakiness of the tetracycline-responsive promoter (tRP) in several commonly used cell lines, resulting in 47- to 328-fold regulation. Furthermore, pSialV also showed efficient regulation in stable cells . [0013] Another disadvantage of the system is its high leakiness from the switched-off TRS. This not only reduces the efficiency of regulation but also prevents the use of the system for cytotoxic gene control.
  • the TRS may have potential immunogenicity even though no anti-tTA antibody has been detected within a 6 month period (Bohl, D., Naffakh, N., and Heard, J.M. (1997). Long-term control of erythropoietin secretion by doxycycline in mice transplanted with engineered primary myoblasts. Na t . Med. 3:299-305, incorporated herein by reference in its entirety) .
  • a gene expression system is thus provided that is suitable not only for use in vitro as a dosage-dependent regulatable gene expression system, but for use ex vivo, in vivo and in gene therapies.
  • the expression system includes a control region including two or more transactivator DNA binding domains that are spaced such that when bound by a transactivators, the transactivators are substantially rotationally aligned about the DNA helix.
  • this control region is operably linked to a promoter to form a regulatable promoter (RP) .
  • the RP may be attached to a polylinker or recombination sequence which is also attached to a polyA signal opposite the RP, thereby producing an expression cassette into which a coding sequence may be inserted for regulatable expression of the coding sequence.
  • the transactivators binding domains, RP or expression cassette may be inserted into a vector for either propagation of the vector or for gene expression in a cell.
  • This system displays high regulation efficiency.
  • a tetracycline-regulatable system including a tRP having two or more tetO sequences operably linked to a promoter
  • this efficiency is seen in both up- regulatory (as with a TET-ONTM system) or down-regulatory (as with a TET-OFFTM system) systems, as well as single or double plasmids .
  • a humanized transactivator such as the tetR-NF- ⁇ B p65 transactivator described below, this system is potentially less immunogenic.
  • One specific embodiment employs 23-29, 33-39 or 43-50, preferably about 26, 36 or 46 base pairs or about 2 , 3 or 4 helical turns between the central bases of each adjacent pair of 8 tetO sequences and, together with a minimal CMV promoter, forms a second generation tRP.
  • This second generation tRP displays maximal expression 5- to 10-fold higher than the original tRP in the context of both up- regulatory and down-regulatory systems.
  • the efficiency of the new tRP is increased by 5- to 10-fold from two orders of magnitude of regulation to three orders of magnitude.
  • the regulatable promoter systems described herein are completely interchangeable with the first generation promoter systems that are commercially available. As such, they are applicable in a large variety of in vi tro, ex vivo and in vivo applications as described in the literature for the first generation systems, including, without limitation: cell and gene therapies; veterinary uses; yeast expression systems; gene function studies; conditional immortalization of primary cells of therapeutic relevance; biocatalysis, fermentation and bioprocessing of therapeutic or other molecules; transgenic plants and animals; high throughput screening applications; functional genomics and target validation; and in plant biosciences, including establishment of disease and pest resistance.
  • Also provided is a method for modulating expression from a regulatable promoter including two or more transactivator binding domains. The method includes the step of modifying the distance between the two or more domains to achieve a desired degree of regulation efficiency, that is to vary the maximal and basal transcription rates .
  • Figure 1 shows the sequences of the 2tetO inserts (SEQ ID NOS: 1-8).
  • the tetO sequence is shown in bold with the central base, G, in italic.
  • the random bases intervening between the 2 consecutive tetO sites is shown in normal font.
  • the sequence intervening between the 2 central bases is underlined. Bps states for base pairs.
  • TRS With regard to the TRS, achieving regulated gene expression is still a cumbersome procedure, requiring several months to establish a stable cell line. In addition, the efficiency of the original TRS is unpredictable and not only depends on the ratio and concentration of the delivered transactivator and reporter plasmids, but also on the site of integration of the plasmids intra-chromosomally. Most reports using the TRS have at best managed to achieve 10- to 100-fold regulation with few studies exceeding this . For in vivo gene regulation, where absolute concentration of tetracycline is subject to individual's pharmacokinetics and pharmacodynamics, 100-fold regulation is less than desirable. In these instances, a 1000-fold regulation is far more practical, with the aim of reaching 100%, 10%, 1% and 0.1% gene regulation.
  • the original TRS also displays relatively high basal leakiness that is a function of the minimal CMV promoter of tRP and it is therefore cell-type dependent (Agha-Mohammadi and Hawkins, 1998) .
  • Several permutations have attempted to reduce this leakiness through the use of an alternative minimal promoter that simultaneously reduced maximal expression (Hoffmann, A., Villalba, M. , Journot, L., and Spengler, D. (1997).
  • the TRS transactivators may have potential immunogenicity. Even though, gene expression and regulation have been followed for more than 6 months, with no detection of anti-tTA antibody (Bohl et al., 1997), both tetR and VP16 domains are foreign and potentially immunogenic.
  • a "second generation tetracycline regulatable system” was constructed and characterized. As discussed in further detail below, the second generation tRPs were devised to reduce the basal expression of previously described pCMV* -1 .mGM-CSF, while enhancing the efficiency of regulation. By removing the upstream sequence of mGM-CSF, plasmid pCMV* -2 .mGM-CSF with reduced basal expression was constructed. Plasmid pCMV* -2 .mGM- CSF, which has the same minimal promoter as tRP, showed approximately 10-fold reduced basal expression compared to pCMV* "1 .mGM-CSF. Plasmid pCMV* -2 .mGM-CSF was selected as a control to represent the activity of the tRP.
  • Tet repressors bound to the TnlO- encoded tet gene control region determined by neutron solution scattering. EMBO J. 8,1257-1263, incorporated herein by reference in its entirety) .
  • a similar unwinding of DNA by tTA can position all the VP16 transactivational domains on the same side of the DNA in 2tetO-26, -36, and -47 plasmids where maximal synergy may occur.
  • results obtained from these experiments substantiate the transactivator synergy model of transcription.
  • transcriptional synergy occurs when closely aligned transactivators cooperate to initiate transcription.
  • the results presented herein support the model of transactivator synergy and further highlight the importance of positions of DNA binding sites in this process.
  • the data obtained can be applied to optimize other existing regulatable systems, such as the progesterone-, ecdysone-, rapamycin-regulatable systems (Agha-Mohammadi and Lotze, 2000) by maximizing transactivator synergy through rotational alignment
  • tTA synergy was augmented through the use of multiple tetO sequences.
  • tTA synergy was progressively enhanced by increasing the number of tetO sites from 2 to 8, resulting in higher efficiencies of regulation.
  • One plasmid, p ⁇ tetO- 36.mGM-CSF had a 5- to 10-fold higher regulation efficiency than the original tRP by attaining 5- to 10-fold higher maximal expression while conserving the basal expression.
  • p8tetO- 36.mGM-CSF consistently displays over a 1000-fold gene regulation.
  • the second generation tRPs Compared to the original TRS, the second generation tRPs also displayed 5- to 10-fold improved efficiencies in 293 cells and NIH 3T3 cell lines (Data not shown) . Similar findings have been noted on using the second generation tRPs in the context of the Tet-on system (See Example 6, below) . Thus, the second generation tRPs overcome the relative inefficiency of the original tRP, especially for in vivo use. In the Examples, below, pCMV *-2 .mGM-CSF was used so that the efficiency of the new second generation tRP can be compared with the original tRP. The observed leakiness of the second generation tRP and the original tRP are similar in these experiments .
  • a number of shortened versions of the minimal tRP promoters were made having reduced basal leakiness while preserving the high efficiency of the second generation tRP.
  • other regulatable systems may benefit from inclusion of more than two DNA transactivator binding domains.
  • An important application of the second generation tetracycline regulatable system is in construction of tetracycline-regulated replication-effective viruses.
  • replication-effective adenoviruses that are controlled through regulation of the El gene of the adenovirus will be of importance in future cancer gene therapy.
  • the tetracycline regulatable system is one of the most valuable tools for controlling gene expression both in vitro and in vivo .
  • This system still suffers from a number of imperfections. These concerns are of exceptional importance in in vivo use of the system where ease of set-up and delivery, high regulation efficiency, and reduced immunogenicity are particularly needed.
  • a second generation of tetracycline- regulated promoters and transactivators were devised.
  • the second generation tRPs display enhanced regulation efficiency by increasing the maximal expression of the promoter by augmenting tTA synergy. These promoters sustain their regulation efficiency in the context of a single positive feedback regulatory vector that presents ease of delivery of the system for in vivo and gene therapy use.
  • second generation transactivators utilizing human transactivational domains should reduce the potential immunogenicity of the original system.
  • nucleic acid comprising in .its most minimal sense two or more transactivator binding domains, such as tetO sequences, separated by a specific number of bases so that transactivators binding adjacent transactivator binding domains are rotationally aligned about the DNA helix.
  • transactivator binding domains such as tetO sequences
  • adjacent tetO sequences that are not bound by transactivator fall on opposite sides of the DNA helix and, when bound by transactivator are approximately aligned at their palindromic centers.
  • a transactivator binding domain is a sequence to which a transactivator or transrepressor can bind and includes, without limitation, tetO sequences, GAL4-DNA binding sequences, ecdysone ECRE sequences and the rapamycin system' s ZFHD1 sequences .
  • a "turn" of the double helix is in reference to the relative rotational relationship between two bases in most DNA forms, with each 10-11.5 base separation between the two bases being a single “turn”, referring to a single 360° rotation of the DNA helix.
  • a single turn is a separation of about 10 bases, 2 turns is about 21 bases and 2 ⁇ - turns is about 26 bases, recognizing that the number of turns is a close approximation because a single turn corresponds roughly to a non-integer 10.4 bases.
  • center nucleotides of adjacent tetO sequences are typically separated by 23 to 29, 33 to 39 or 43 to 50 bases, more typically by 25 to 27, 35 to 37 or 45 to 47 bases, and as provided in the Examples below, by 26, 36 or 46 bases .
  • tetO sequence it is meant a tetracycline operator sequence.
  • tetO sequences include Al, A2, Bl, B2, CI, C2, Dl, D2, El and E2 tetO sequences (native tetO sequences) and operable mutants thereof.
  • tetO sequences as described, for instance in U.S. Patent No 6,138,954, Fig 5, incorporated herein by reference, have palindromic features and typically have a single "central nucleotide” that is the center of the palindromic sequence .
  • Mutants of the tetO sequence are known and are limited only by their ability to bind to a corresponding tetracycline repressor (tetR) protein either in native form or as a fusion protein with a eukaryotic transactivator peptide, such as herpes simplex VP16, Epstein- Barr virus (EBV) rta and human NF- B p65 transactivators (tTA and rtTA) .
  • tetO sequences are described in Sizemore C, Wissmann A., Gulland U. and Hillen W. (1990). Quantitative analysis of TnlO Tet repressor binding to a complete set of tet operator mutants. Nucl . Acid Res . 18(10) :2875-2880.
  • a "fusion protein” refers to two or more polypeptides that are operatively linked and typically, but not necessarily, form a single polypeptide chain. Further, each operatively linked polypeptide in the fusion protein can serve its intended function and the fusion peptide can serve its intended function.
  • Operative mutants of tetO sequences include native tetO sequences having insertions, deletions and substitutions that do not result in the inability of tetR, tTA or rtTA proteins to bind the tetO mutant either in the absence of tetracycline (a Tet-off system) , as in the case of the tetR and tTA proteins, or in the presence of tetracycline (a tet-on system), as is the case with rtTA proteins.
  • transactivator DNA binding sequences are known and transactivators that bind those sequences also have been described. These include: the HSV VP16-GAL4 DNA-binding domain/mutant progesterone receptor (PR-LBD) transactivator of the progesterone-regulatable system; a heterodimeric complex of VpECR (a fusion of VP16, the N-terminal truncation of a mutant ecdysone receptor and the DNA-binding domain of the glucocorticoid receptor) and the retinoid X receptor in the ecdysone-regulatable system; and the ZFHD1 (DNA binding unit)- FKBP12 chimera in combination with the FRAP/NF- B p65 chimera in the rapamycin-regulatable system (see, e.g., Agha-Mohammadi and Lotze, 2000) .
  • PR-LBD DNA-binding domain/mutant progesterone receptor
  • tetR is a protein that specifically binds a tetO sequence in the absence of tetracycline .
  • Native tetR proteins are specific to each native tetO sequence .
  • a tetR that binds the A-type tetO sequence does not necessarily bind B-, C-, D- and E-type tetO sequences.
  • a tetracycline transactivator (tTA) is a fusion protein between a tetR sequence and a eukaryotic transactivator.
  • the tetR sequences in the fusion protein are substantially complete in that they include DNA binding- and tetracycline-binding domains as well as all peptide sequences necessary for tetracycline-regulatable binding to the tetO sequences. In the presence of tetracycline, tTA does not bind its corresponding tetO sequence.
  • the tetR portion of the tTA fusion protein minimally includes all sequences necessary for binding of the tTA to the tetO sequence in the absence of tetracycline and the release (non-binding) of tTA from the tetO sequence in the presence of tetracycline.
  • the rtTA fusion protein contains the same transactivational domain as the tTA fusion protein, but contains a mutated tetR portion that, as opposed to the tTA protein, binds its corresponding tetO sequence in the presence of tetracycline and does not bind its corresponding tetO sequence in the absence of tetracycline.
  • tTAs such as that encoded by pUHD 15-1
  • rtTAs such as that encoded by pUHD 17-1 are provided in Example 1 of U.S. patent No.6, 136, 954 (Columns 45 and 46, incorporated herein by reference) .
  • transrepressors may be useful in the regulatable systems described herein.
  • Deuschle, U., Meyer, W.K.H., and Thiesen, H.J. (1995) describe a fusion protein consisting of a tetR unit and the KRAB repressor domain of the human Koxl zinc finger protein. Therefore, in addition to the transactivator fusion proteins, transrepressor proteins may be used in the optimized regulatable systems described herein.
  • the nucleic acid described herein minimally includes 2 and typically includes 2-8 tetO sequences distanced from each other as described above so no adjacent tetO central nucleotides are in rotational alignment when the nucleic acid is in B form. Typically, each adjacent pair of tetO sequences are spaced from each other the same distance as other adjacent pairs of tetO sequences in the same nucleic acid sequence.
  • the nucleic acid may be only long enough to include the plurality of tetO sequence, optionally including a linker or a recombination sequence at its ends, and, therefore, is useful as an insert for a gene expression system.
  • the insert would be placed an appropriate distance from and location with respect to a promoter so that it is operably linked to that promoter.
  • the addition of linkers or recombination sequences to the ends of the insert would facilitate attachment of the insert to other regulatory and coding sequences.
  • a linker is a short nucleic acid sequence that includes a restriction site that typically is not present in the insert and are typically are restriction sites for rare- cutting restriction endonucleases that are single- or dual- cutters for the sequence to which the insert is to be attached.
  • a polylinker is a linker having more than one restriction site.
  • a recombination sequence is a sequence that when used with particular cloning systems, can direct by a recombination event insertion of a nucleic acid flanked by two recombination sequences into another nucleic acid containing a co-reacting recombination sequence or sequences, such as a CRE-LoxP system (for example, the CREATORTM cloning system commercially available from Clontech and GATEWAY system from Invitrogen) .
  • a CRE-LoxP system for example, the CREATORTM cloning system commercially available from Clontech and GATEWAY system from Invitrogen
  • operably linked means that elements that are connected in a manner such that each element can serve its intended function and the elements, together can serve their intended function.
  • operatively linked means that a first regulatory element or coding sequence in a nucleotide sequence is attached and oriented in relation to a second regulatory element or coding sequence in the same nucleic acid so that the first regulatory element or coding sequence operates in its intended manner in relation with the second regulatory element or coding sequence.
  • a tetO sequence is operatively linked to a promoter to form a tRP that when incorporated into a complete gene, including operably linked tetO sequences, a promoter, a coding sequence and a polyadenylation (polyA) signal, the tetO sequences can be used to control expression of the coding sequence in the presence of tTA and rtTA proteins .
  • a promoter is operably linked to a coding sequence to promote transcription of that coding sequence.
  • a regulatory element is a nucleotide sequence that controls expression of, a coding sequence, alone, or in combination with other nucleotide sequences or trans factors, such as repressor proteins like tetR, activator proteins, such as tTA and rtTA, polymerases and polymerase complex proteins such as initiator proteins.
  • Regulatory elements include, without limitation, operators, enhancers, promoters and polyA signals.
  • sufficient regulatory elements to control gene expression it is meant that a nucleic acid includes any operatively linked regulatory elements needed for the intended expression of the gene, typically including minimally a promoter and a polyA signal, and, in the context of the present disclosure, tetO sequences.
  • a coding sequence is a sequence that either encodes a protein, therefore including an open reading frame or a functional RNA, such as, without limitation, an anti- sense RNA or a ribozyme.
  • the coding sequence may include one or more intron sequences.
  • the nucleic acid described herein typically contains a promoter operably linked to the transactivator binding domains to form a regulatable promoter (RP) , or tRP when the domains are tetO sequences.
  • RP regulatable promoter
  • a "promoter” is a DNA sequence that determines the site of transcription initiation for an RNA polymerase.
  • the expression system described herein includes sequences for binding a transactivator or transrepressor protein, such as the tTA or rtTA proteins, that controls certain promoters
  • a transactivator or transrepressor protein such as the tTA or rtTA proteins
  • the only limitations places on the provided definition of "promoter” is that the “promoter” is selected to be responsive to transcriptional regulation by a the transactivator or transrepressor when that protein is bound to the transactivator binding domains operably linked to the promoter. This requirement, in many cases, is not very stringent in that many known transactivators can enhance transcription from many different promoters.
  • herpes simplex virus VP-16, EBV rta and human NF- ⁇ B p65 transactivators enhance transcription from many known eukaryotic promoters, including viral promoters, notably the CMV promoter as is described in the literature with regard to tetracycline-responsive systems, and as is described herein.
  • a promoter can be, without limitation, constitutive, tissue-specific, or otherwise controllable.
  • promoters include, without limitation, the CMV, HSV-Tk, human ⁇ -actin, muscle creatinine kinase (MCK) , amylase and albumin promoters.
  • MCK muscle creatinine kinase
  • any promoter can be tested readily for its e fectiveness in the tetracycline- responsive expression system described herein by substitution for the minimal CMV promoter described herein.
  • Modified promoters also may be used, including insertion and deletion mutation of native promoters and combinations or permutations thereof.
  • One example of a modified promoter is the "minimal CMV promoter" the sequence of which is provided in Example 5 (SEQ ID NO: 21) , below.
  • An "expression cassette" including the above- described RP including a promoter, such as a viral promoter, for instance a CMV or CMV minimal promoter, and a polyA sequence.
  • a linker preferably a polylinker containing multiple single-cut restriction sites, or a recombination sequence, such as LoxP, is placed between the promoter and polylinker facilitating insertion of, and thereby operable linkage of any coding sequence to the promoter and polyA site.
  • the expression cassette typically is propagated as an autonomously replicating genetic unit, most typically as a plasmid, more typically as a ColEl plasmid containing an E. coli replication origin and one or more selectable markers, for instance antibiotic resistance genes, for selection in bacteria and/or in eukaryotic cells.
  • the above-described RP may be in the form of a gene in which the RP is operably linked to a coding sequence and a polyA signal, may be operably linked to 1) sequence (s) that permit packaging of the gene into a virus particle, 2) sequence (s) that permit autonomous eukaryotic propagation of the plasmid, such as viral origins of replication and/or 3) sequence (s) that permit or facilitate integration of the gene containing the tetO/promoter sequence into a eukaryotic genome.
  • viral vectors examples include: 1) the tetracycline-regulatable gene or expression cassette flanked by two retroviral LTR sequences (see, for example, S. Agha-Mohammadi and Lotze, M.T. (1998). "Regulatable Systems: Applications in Gene Therapy and Replicating Viruses" J. Clin . Investiga tion 105 (9) : 1177-1183, incorporated herein by reference in its entirety; U.S. Patent No. 6,133,027, specifically columns 17 to 32 thereof, which is incorporated herein by reference; and U.S. patent No.
  • Retroviral and Adenoviral tetracycline- regulatable expression systems are commercially available from Clontech.
  • the nucleic acid also may contain a gene for the production of the tTA or rtTA protein, most preferably including a sequence encoding the tTA or rtTA protein operably linked to a promoter which, in turn, is operably linked to the tetO sequences, but oriented in a direction opposite that of the first tetracycline-regulatable expression cassette or gene.
  • a gene for the production of the tTA or rtTA protein most preferably including a sequence encoding the tTA or rtTA protein operably linked to a promoter which, in turn, is operably linked to the tetO sequences, but oriented in a direction opposite that of the first tetracycline-regulatable expression cassette or gene.
  • This is, in one embodiment, a second generation version of the single self-contained vector tetracycline-regulatable expression systems described in the literature, such as in S. Agha-Mohammadi and Lotze, M.
  • This second generation version substitutes the novel second generation tRP described herein for the first generation tRP.
  • the tetO sequences bind tTA or rtTA fusion proteins, which control expression bi-directionally from both the tTA or rtTA gene and the first gene described above containing any coding sequence. Similar constructs may be made to produce transactivators or transrepressors for regulatable systems other than the TRS .
  • the coding sequence for tTA or rtTA is substituted with a different coding sequence, permitting bi-directional expression of two different genes, such as a marker and another gene that cannot be easily detected.
  • Such vectors, using the first generation tRP are commercially available from Clontech, and are designated pBI TET vectors.
  • Transactivators, such as tTA and rtTA must be provided in these systems either by co-transfection of a plasmid containing the tTA or rtTA genes, or by other means.
  • the transactivator or transrepressor proteins also may be provided by a gene that has been established in a cell line.
  • Cell lines transformed with the tTA or rtTA genes are available commercially from Clontech. These cell lines are especially useful when a biocatalyst or producer cell line is desired.
  • Cells transformed by the transactivators can be transformed with a gene under control of the second generation tRP described herein to produce a desired biocatalyst or to produce a desired gene product, for instance in biopharmaceutical production.
  • the second-generation tetracycline-regulatable gene expression systems described herein may be included in a variety of vector systems and may be used in any application that the first generation systems may be used.
  • the expression cassette, gene or single-vector embodiments may be placed in any vector, such as, without limitation, plasmids, phagemids, cosmids, phage and viral vectors, insect vectors, yeast vectors (such as a yeast artificial chromosome), plant vectors (such as Ti plasmids) and combinations thereof as are known in the art.
  • transgenic or homologous or enzyme-assisted recombination non-human animals such as mice or farm animals, including goats, sheep, pigs, or cows
  • plants are known and conventional in the art and are described, for instance in U.S. Patent No. 5,589,362, columns 15 and 16, incorporated herein by reference.
  • a "cell” may be any cell in which the nucleic acid can either be propagated or can be used to introduce a foreign gene into the cell to either temporarily or permanently transform the cell.
  • the nucleic acid is typically propagated in bacterial cells, most commonly as a ColEI plasmid in an E. coli cell.
  • bacterial cells most commonly as a ColEI plasmid in an E. coli cell.
  • other systems for propagation of vectors are known, including without limitation other bacteria, yeast, insect cells and mammalian cells.
  • Mammalian cells may be preferred if the vector is a viral vector such as an adenovirus, a retrovirus or an adeno- associated virus.
  • the cells to be transformed, or transformed cells typically are eukaryotic cells and can vary according to the desired application.
  • These cells include: 1) cultured cells, such as HeLa cells optionally transformed with transactivator or transrepressor gene, such as a tTA or rtTA gene, typically for use in research, bioconversion and biocatalysis applications or ex vivo uses, such as in therapeutic applications; 2) cells in vivo, typically for therapeutic applications and in vivo cells transformed with the nucleic acid either by delivery to the organism or by somatic transformations, including transgenic or recombination methods, in which case, the cells range from non-mammalian cells, such as, plant, insect or non-mammalian vertebrate cells to mammalian cells.
  • non-mammalian cells such as, plant, insect or non-mammalian vertebrate cells to mammalian cells.
  • a method for preparing an expressible gene that includes the step of ligating and thereby operably linking a nucleic acid including a coding sequence to a nucleic acid including a second generation tRP as described herein.
  • a method for modulating expression from a regulatable promoter including two or more transactivator binding domains includes the step of modifying the distance between the two or more domains to achieve a desired degree of regulation efficiency, that is to vary the maximal and basal transcription rates.
  • a method for expressing a gene in a cell includes the steps of: introducing a nucleic acid into the cell, the nucleic acid comprising a second generation RP, such as the tRP described herein operably linked to a coding sequence, and expressing a transactivator protein, such as a tTA or rtTA protein, in the cell, such that expression of the coding sequence is activated either when the cell is contacted with an inducer, such as tetracycline, or an analog thereof, or by the removal of the inducer from contact with the cell.
  • the nucleic acid may be introduced into the cell by any method, including, without limitation, viral transduction, liposome transfection, calcium phosphate precipitation, DEAE-dextran transformation, particle bombardment, microinjection, and endocytosis .
  • tetracycline analogs may be as useful, or more useful than tetracycline for the purpose of binding tetR, or transactivator or transrepressor derivatives thereof.
  • doxycycline may be preferred to tetracycline in its use in binding to tetR or derivatives thereof.
  • Other useful pharmaceutically acceptable tetracycline analogs include: chlortetracycline, oxytetracycline, demethylchloro- tetracycline, methacycline, doxycycline and minocycline.
  • a method for controlling expression of a tetracycline-regulatable gene including the step of contacting a cell containing a nucleic including the tetracycline- regulatable gene with one of chlortetracycline, oxytetracycline, demethylchloro-tetracycline, methacycline, doxycycline and minocycline.
  • DCs dendritic cells
  • DCs may be isolated by known methods and transformed with a gene as described above.
  • DCs are capable of migrating into tissues, taking up antigen and then migrating into lymph nodes. This property confers on them an ability to deliver cytokines, genes, drugs or prodrugs to tissues.
  • DCs may be transformed ex vivo with a regulatable gene as described herein and re-introduced into a patient in order to target delivery of the regulatable gene to a desired location.
  • DCs target sites of inflammation, which may be a condition of the patient, or which may be induced by injury or other agents.
  • DCs chronic inflammation including cancer, chronic viral disease, autoimmunity, or allograft rejection may be accessible to such approaches.
  • Homing of DCs also may be enhanced by expression on the surface of the dendritic cell of a homing receptor or "addressin" specific for tissue or organs.
  • An addressin may be generated by phage-display screening processes as are known in the art and the sequences encoding the addressin might be expressed from a second transformed gene, including from a second coding sequence in the above-described bi-directional tRP system.
  • the gene to be inserted into the dendritic cells may be one of any class of gene that would be useful, including: cytokines for targeted secretion; antigen for targeted presentation as, for instance, a vaccine; a co- stimulatory surface-expressed molecule; an enzyme that can convert an inactive prodrug or other inactive compound to an active drug or compound for targeted drug delivery; and a system including a dendritic cell that is pre-loaded with a drug in vesicles (for instance, by liposome or exosome delivery) and which contains an inducible gene that causes release of the drug after the dendritic cell homes to its target site.
  • a non-leaky regulatable promoter such as the promoter of Example 5, below, may be preferred.
  • Plasmids and DNA manipula tion [0061] The plasmid pCMV *-1 .mGM-CSF has been described before (Agha-Mohammadi et al . , 1997). This plasmid contains about 50 bases upstream of the mGM-CSF cDNA and downstream to the minimal CMV promoter of tRP.
  • the plasmid pUHD 15-1 (Clontech) contains the tTA transactivator gene transcribed from the CMV IE promoter (Gossen and Bujard, 1992) .
  • Plasmid p8tetO-16.mGM del Rl-SacII has a deletion of sequence corresponding to bases 439 to 449 of pUHD 10-3 (Gossen and Bujard, 1992). This deletion removes 10 bases of the distal end of the minimal CMV promoter.
  • the plasmid p8tetO-16.mGM del Rl-Bbsl has a deletion of sequence corresponding to bases 414 to 449 of pUHD 10-3. This deletion removes 35 bases of the distal end of the minimal CMV promoter.
  • Plasmid p8tetO-16.mGM del Kpnl-Stul has a deletion of sequence corresponding to bases 304 to 336 of pUHD 10-3. This deletion removes 32 bases upstream of the TATA box.
  • the oligonucleotides 5' TCGAGCTCCCTATCAGTGATAGAGAAGGTAC 3' (SEQ ID NO: 9) and 5' CTTCTCTATCACTGATAGGGAGC 3' (SEQ ID NO: 10) were annealed and ligated into Xhol/Kpnl digested pCMV* "2 .mGM-CSF.
  • the annealed oligonucleotide corresponds to the sequence of a single tet0 2 with Xhol and Kpnl overhangs.
  • Plasmids, p2tetO- 21.mGM-CSF, p2tetO-26.mGM-CSF, p2tetO-31.mGM-CSF, p2tetO- 36.mGM-CSF, p2tetO-42.mGM-CSF, p2tetO-47.mGM-CSF, p2tetO- 52.mGM-CSF, and p2tetO-57.mGM-CSF were constructed by placing a second tet0 2 oligonucleotide, between Xhol and Aatll sites, at 3, 8, 13, 18, 24, 29, 34, and 39 bases away from the first tet0 2 element, respectively.
  • Plasmids p4tetO-26.mGM-CSF, p4tetO-36.mGM-CSF, and p4tetO- 47.mGM-CSF were constructed by digesting the respective 2tetO plasmids with Aatll and BamHI to remove the 2tetO sites/minimal promoter/mGM-CSF cassette.
  • Plasmid p4tetO-26.mGM-CSF was constructed by placing blunt-ended 2tetO-26/minimal promoter/mGM-CSF insert into blunt-ended Kpnl/BamHI cut p2tetO-26.mGM-CSF backbone and confirmed by restriction digest and sequencing for the plasmid with 4 tetO sites each separated by 8 bases by sequencing.
  • the 2tetO-47/minimal promoter/mGM- CSF insert was ligated into Kpnl/BamHI cut p2tetO-47.mGM-CSF backbone via a random linker (5' CCCGGAATCGAGTCATCGACGT 3' (SEQ ID NO: 13) and 5' CGATGACTCGATTCCGGGGTAC 3' (SEQ ID NO: 14)) with restriction overhangs for Kpnl and Aatll.
  • a random linker 5' CCCGGAATCGAGTCATCGACGT 3' (SEQ ID NO: 13) and 5' CGATGACTCGATTCCGGGGTAC 3' (SEQ ID NO: 14)
  • Plasmid p6tetO-36.mGM-CSF was constructed by inserting the 2tetO-36/minimal promoter/mGM-CSF cassette into the Kpnl/BamHI digested p4tetO-36.mGM-CSF using linker-18.
  • p8tetO-36.mGM-CSF was constructed by inserting the 2tetO-36/minimal promoter/mGM-CSF cassette into the Kpnl/BamHI digested p6tetO-36.mGM-CSF using linker-18.
  • Plasmid PFRVII was constructed by removing tTA from pCI.tTA.neo (Agha- Mohammadi and Hawkins, 1998) with Aatll and Drain. The blunt-ended fragment was then ligated into the blunt-ended, alkaline phosphatase-treated Aatll site of p8tetO-36.mGM-CSF. Plasmids with tTA and mGM-CSF oriented in opposite directions were selected.
  • pCMVtetR-Rta To construct pCMVtetR-Rta, pUHD 15-1 was cut with EcoRI and Avail and a 624 base pair fragment representing tetR was isolated. This fragment, together with a synthetic linker, were ligated into EcoRI/BamHI cut pUHD 15-1 to form pCMVtetR-Bsu36l .
  • the synthetic linker comprising of 5' GTCCCTAAGGTCG 3' (SEQ ID NO: 15) and 5' GGATTCCAGCCTAG 3' (SEQ ID NO: 16), placed a unique Bsu36I restriction site distal to tetR.
  • the plasmid pMH212 (a gift of Mary Hardwick, Hardwick, J.M., Tse, L., Applegren, N., Nicholas, J., and Veliuona, M.J. (1992) .
  • the Epstein-Barr virus R transactivator (Rta) contains a complex, potent activation domain with properties different from those of VP16. J. Virol. 66,5500-5508) was digested with Bsu36l and Bglll to remove the C-terminal 520- 605 amino acids of Rta. This fragment was inserted in frame with the coding sequence of tetR into Bsu361/BamHI cut pCMVtetR-Bsu36I.
  • Plasmid pCMVtetR-p65 281 was constructed by removing amino acids 281-551 of NF- ⁇ B p65 (Ballard, D.W. et al. (1992).
  • the 65-kDa Subunit of human NF- ⁇ B functions as a potent transcriptional activator and a target for v-Rel- mediated repression.
  • Structural and Functional Analysis of the NF- ⁇ B p65 C terminus J. Biol. Chem. 269 (41) :25613-25620) of pCMV4-p65 (kindly provided by W Greene) with EcoRI and BamHI. This was then ligated into
  • Plasmid pCMVtetR-p65 313 was constructed by removing amino acids 313-551of NF- ⁇ B p65 with BspHI and BamHI. This was then ligated into Bsu361/BamHI cut pCMVtetR-Bsu36I via a serine/glycine/glycine linker using the annealed oligonucleotides 5' TAAGTGGCGGGAT 3' (SEQ ID NO: 19) and 5' CATGATCCCGCCAC 3' (SEQ ID NO: 20) with overhangs for Bsu361 and BspHI. The sequences of all constructed plasmids were verified by automated sequencing.
  • HeLa cells were maintained in 10% fetal bovine serum in Dulbecco' s modified Eagle's medium. Transfection of purified plasmid DNA into the cell lines was carried out using a modified calcium phosphate precipitation protocol. To ensure similar transfection efficiencies among wells transfected with the same plasmid, a single precipitate was prepared and ' divided among the wells. To monitor the efficiency of the transfections, ⁇ -galactosidase expression from pCHl 10 (Pharmacia) was assessed as described previously (Agha-Mohammadi and Hawkins, 1998). Generally the difference between the number of cells stained was within 1.0- to 1.3- fold between the wells for a cell-type.
  • Plasmid pCMV *"2 .mGM-CSF which contains the native tRP was then used to optimize tTA transactivational synergy by repositioning tetO arrangement.
  • the effect of distance and position of tandem tetO sequences on tTA transactivational synergy was evaluated by varying the minimum distance between the central bases of 2 consecutive tetO sequences from 2 to 5 helical turns at turn increments.
  • HeLa cells in 6-well plates were co-transfected with 1.5 ⁇ g of pCMV *"1 .mGM-CSF, pCMV *-2 .mGM-CSF, p2tetO-21.mGM- CSF, p2tetO-26.mGM-CSF, p2tetO-31.mGM-CSF, p2tetO-36.mGM-CSF, p2tetO-42.mGM-CSF, p2tetO-47.mGM-CSF, p2tetO-52.mGM-CSF, or p2tetO-57.mGM-CS, and 1.5 ⁇ g of pUHD 15-1, together with 0.5 ⁇ g of pCHHO as a control for assessing transfection efficiency. Experiments were repeated 4 times to establish a pattern.
  • the maximal expression and thus efficiency of the 2tetO promoters is dependent on the separation distance and position of the 2 consecutive tetO sites.
  • the constructed plasmids displayed comparable basal expressions.
  • Plasmid p2tetO-21.mGM-CSF has 2 tetO sites separated by 3 base pairs. The central base of each palindromic tetO site is therefore positioned exactly on the same side of the DNA, considering a 10.4 base for each helical turn of B-DNA and a 9 base flank on each side of the central base of tetO.
  • This plasmid displayed the lowest maximal expression which probably reflects hindrance to the attachment of tTA molecules to the adjacent tetO sites. Maximal expression is best achieved when the distance between centers of the 2 tetO sites ranged from 26 to 52 bases. Within this range, maximal expression varies more significantly with the positional relations of the 2 tetO sites, rather than simply the distance between the sites.
  • Plasmids 2tetO-26, 2tetO-36, and 2tetO-47 display the highest maximal expression and the most efficient regulation in response to Dox.
  • the central bases of the 2 tetO sites are separated by 2 ⁇ _, 3 ⁇ *$., and 4 ⁇ helical turns and are therefore positioned on opposite sides of the DNA.
  • the efficiency of plasmids 2tetO-31, 2tetO-42, and 2tetO-52 are similar and as a group, uniformly lower than those of 2tetO-26, 2tet036, and 2tetO-47 plasmids.
  • Plasmids p2tetO-26.mGM-CSF, p2tetO-36.mGM-CSF, and p2tetO-47.mGM-CSF consistently displayed about 1000-fold regulation efficiencies which is about 2-fold higher than that achieved with pCMV *-2 .mGM-CSF) . This appears to be the highest regulation ever obtained when equal ratios of transactivator and reporter plasmids are co-transfected.
  • Plasmid pCMV *-2 .mGM-CSF has 7 tetO sequences with tetO center-center separations of 41 bases pairs, and thus resemble p2tetO-42.mGM-CSF in having tetO central bases that are positioned on the same side of the DNA.
  • HeLa cells in 6-well plates were co-transfected with 1.5 ⁇ g of p2tetO-21.mGM-CSF, p2tetO-26.mGM-CSF, p2tetO- 31.mGM-CSF, p2tetO-36.mGM-CSF, p2tetO-42.mGM-CSF, p2tetO-
  • Example 2 Second generation tetracycline- egula ted promoters [0075]
  • the results obtained with the 2tetO plasmids indicated that the maximal expression of tRPs can be enhanced by modifying the arrangement of the tetO sequences .
  • To further enhance the regulation efficiency of the new tRPs we attempted at increasing the maximal expression of 2tetO-26, 2tetO-36, and 2tetO-47 vectors by using multiple tetO sites. Since the efficiencies of 2tetO-26, 2tetO-36, and 2tetO-47 were not significantly different in several repeated studies, plasmids with 4 tetO elements were constructed based on these vectors. In these promoters, center-center separation of consecutive tetOs were set at 26, 36, or 46 random base pairs, respectively. The plasmids were compared relative to pCMV " 2 .mGM-CSF in co-transfection studies.
  • HeLa cells in 6-well plates were co-transfected with 1.5 ⁇ g of pCMV *-1 .mGM-CSF, p4tetO-26.mGM-CSF, p4tetO- 36.mGM-CSF, or p4tetO-47.mGM-CSF and 1.5 ⁇ g of pUHD 15-1, together with 0.5 ⁇ g of pCHHO as a control for assessing transfection efficiency.
  • HeLa cells in 6-well plates were co-transfected with 1.5 ⁇ g of pCMV *-2 .mGM-CSF, p2tetO-36.mGM-CSF, p4tetO- 36.mGM-CSF, p6tetO-36.mGM-CSF, or p8tetO-36.mGM-CSF, and 1.5 ⁇ g of pUHD 15-1, together with 0.5 ⁇ g of pCHllO as a control for assessing transfection efficiency.
  • the final mGM-CSF value represents the average of 6 samples. The standard error of each average was consistently less than 10% of the final value. Experiments were repeated several times to establish a pattern.
  • Example 3 Second generation tetracycline-regula ted promoter functions efficiently as a single positive feedback regulatory vector
  • PFRS positive feedback regulatable system
  • both tTA and mGM-CSF will be produced at basal levels . Subsequently, tTA engages in a positive feedback loop up-regulating its promoter bidirectionally.
  • the system offers increased sensitivity to tetracycline repression by a dual mechanism, i.e. terminating tTA transcription and causing its inactivation.
  • the results obtained from these studies indicate that the second generation tRP maintains its low basal expression and high regulation efficiency within the context of a positive feedback regulatable vector, as shown in Figure 2.
  • the PFRV II plasmid attains regulation efficiencies close to 900-fold in optimized experiments; a level several fold higher than the original co-transfected TRS.
  • the single PFRV II plasmid is a simple and highly efficient regulatable vector that can be conveniently delivered via viral or non-viral means .
  • the data for Figure 2 was generated as follows. HeLa cells in 6-well plates were co-transfected with 1 ⁇ g of PFRV II and 1 ⁇ g of pBR322, 1 ⁇ g of pCMV *"1 .mGM-CSF and 1 ⁇ g of pUHD 15-1, or 1 ⁇ g of p8tetO-36.mGM-CSF and 1 ⁇ g of pUHD 15-1, together with 0.5 ⁇ g of pCHllO as a control for assessing transfection efficiency.
  • the final mGM-CSF value represents the average of 6 samples. The standard error of each average was consistently less than 10% of the final value. The experiment was repeated 2 times to establish a pattern.
  • Example 4 Humanized tetracycline-regulated transactivators: tetR-p65 [0083]
  • the TRS may elicit an immune response that limits gene expression. Even though, gene expression and regulation have been observed for over 6 months, with no detection of anti-tTA antibody (Bohl et al . , 1997), both tetR and VP16 domains are foreign and potentially immunogenic. To reduce this immunogenicity, we have replaced the VP16 domain of tTA with the C-terminal 286- 551 and 313-551 amino acids of human NF- ⁇ B p65 protein and the efficiencies of these chimera were compared with that of tTA in co-transfection studies.
  • tetR-p65 281 and pCMV. tetR-p65 313 functioned efficiently as tetracycline-regulated transactivators, reaching regulation efficiencies comparable with that of tTA, as shown in Table 5, below. Similar observations have been reported in the context of the rapamycin-dependent transactivator of RRS (Rivera, V.M., Clackson, T., Natesan, S., Pollock, R., Amara J.F., Keenan, T., Magari, S.R., Phillips, T., Courage, N.L., Cerasoli, Jr., F., Holt, D.A., and Gil an, M. (1996) .
  • the new humanized tetracycline-regulated transactivator should reduce the potential immunogenicity of tTA, although it will not prevent an immune response against the tetR domain.
  • HeLa cells in 6-well plates were co-transfected with using 1 ⁇ g of pCMV.tetR-p65 281 , pCMV. tetR-p65 313 , or pUHD 15-1 and 1 ⁇ g of p8tetO-36.mGM-CSF, together with 0.5 ⁇ g of pCHHO as a control for assessing transfection efficiency.
  • the final mGM-CSF value represents the average of 6 samples. The standard error of each average was consistently less than 10% of the final value. The experiment was repeated 2 times to establish a pattern.
  • Example 5 Reducing the basal leakiness of the minimal CMV promoter in the context of a second generation tetracycline- regula table promoter
  • the leakiness of the tetracycline regulatable promoter essentially reflects the basal expression of the minimal CMV promoter
  • various approaches are being explored to reduce this leakiness.
  • the leakiness and maximal expression can be reduced either by using alternative minimal promoter, such as the promoter of thymidine kinase, or by reducing the minimal CMV promoter even further.
  • the latter approach was explored by constructing a number of vectors that have deletions within the CMV promoter.
  • the following sequence provides the first 500 bases of pUHD 10-3 (Gossen and Bujard, 1993) .
  • Bases 1 to 300 of the sequence include 7 tetO elements (bold, with central G underlined) .
  • the TATA box is situated between 341 to 347
  • the minimal CMV IE promoter as it is know in the art is defined by bases 304 to 449. Targeted deletions in this sequence were made with the object to produce a promoter with lower maximal and basal expressions without compromising the fold regulation.
  • Plasmid p8tetO-16.mGM del Rl-SacII has a deletion of sequence corresponding to bases 439 to 449 of pUHD 10-3.
  • the plasmid p8tetO-16.mGM del Rl-Bbsl has a deletion of sequence corresponding to bases 414 to 449 of pUHD 10-3.
  • Plasmid p8tetO-16.mGM del Kpnl-Stul has a deletion of sequence corresponding to bases 304 to 336 of pUHD 10-3.
  • p ⁇ tetO- 16.TATAII .mGM. has a micro CMV promoter extending from 6 bases upstream of the TATA box to 36 bases downstream of TATA
  • ND Undetectable.
  • HeLa cells in 6-well plates were co-transfected with 1 ⁇ g of p8tetO-16-TATA.mGM or p8tetO-16.mGM and 1 ⁇ g pUHD 15-1 together with 0.5 ⁇ g of pCHllO as a control for assessing transfection efficiency.
  • the final mGM-CSF value represents the average of 6 samples. The standard error of each average was consistently less than 10% of the final value.
  • the second generation tetracycline-regulated promoter functions well both in the context of a down- regulatory and up-regulatory systems, as shown in Table 7, below. As in the case of the down-regulatory system, the second generation tetracycline-regulated promoter also produces higher maximal expression and thus a higher degree of regulation in the context of the up-regulatory system (when using rtTA) .
  • HeLa cells in 6-well plates were co-transfected with 1 ⁇ g of p2tetO-36.mGM, p4tetO-36.mGM, p6tetO-36.mGM, or p8tetO-36.mGM and 1 ⁇ g pUHD 17-1 (Gossen et al., 1995) together with 0.5 ⁇ g of pCHllO as a control for assessing transfection efficiency.
  • the final mGM-CSF value represents the average of 5 samples . The standard error of each average was consistently less than 10% of the final value.

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Abstract

L"invention concerne des promoteurs régulés (PR) et des transactivateurs hautement efficaces. Les PR contiennent 2-8 domaines de liaison de transactivateurs positionnés d"une manière optimisée les uns par rapport aux autres. Dans un système régulable par tétracycline, le PR présente un rendement de régulation amélioré de 5 à 10 fois par maintien d"une expression maximale élevée tout en offrant une inétanchéité basale réduite de 5 à 10 fois. Dans des études transitoires, ces nouveaux promoteurs présentent une régulation génique supérieure à 900 fois à un rapport de 1:1 entre le transactivateur et le plasmide reporter. De plus, ces promoteurs préservent leur rendement de régulation dans le contexte d"un seul vecteur régulateur à retour positif présentant une facilité d"apport du système pour de nombreuses utilisations, notamment pour une utilisation in vivo et en thérapie génique. Enfin, l"invention concerne des transactivateurs humanisés, par exemple un transactivateur à tétracycline utilisant le domaine de transactivation humain de la protéine NF-λB p65 fusionné à un répresseur de tétracycline (tetR) fonctionnant aussi efficacement que le transactivateur régulé par tétracycline (tTA) et devrait réduire l"immunogénicité potentielle du tTA original.
PCT/US2001/031138 2000-10-03 2001-10-03 Systeme d"expression de genes regulables a haut rendement WO2002028168A1 (fr)

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WO2004109018A1 (fr) 2003-06-06 2004-12-16 Pacheco Pedro Alvares Ribeiro Portique possedant une precontrainte a reglage automatique
WO2017036565A1 (fr) * 2015-09-04 2017-03-09 Universita' Degli Studi Di Padova Procédé de production de cellules souches somatiques
CN107881174A (zh) * 2017-11-22 2018-04-06 张宝会 一种翻译水平基因表达调控的方法及应用
WO2020032057A1 (fr) * 2018-08-07 2020-02-13 Modalis Therapeutics Corporation Nouvel activateur de transcription
RU2800921C2 (ru) * 2018-08-07 2023-08-01 Модалис Терапьютикс Корпорейшн Новый активатор транскрипции

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US5922549A (en) * 1993-05-27 1999-07-13 Boehringer Ingelheim International Gmbh Process for screening substances having a modulating effect on an interleukin-5 receptor mediated cellular signal transmission pathway
US5968773A (en) * 1997-11-14 1999-10-19 Heddle; John A. System and method for regulation of gene expression

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US5922549A (en) * 1993-05-27 1999-07-13 Boehringer Ingelheim International Gmbh Process for screening substances having a modulating effect on an interleukin-5 receptor mediated cellular signal transmission pathway
US5888981A (en) * 1993-06-14 1999-03-30 Basf Aktiengesellschaft Methods for regulating gene expression
US5968773A (en) * 1997-11-14 1999-10-19 Heddle; John A. System and method for regulation of gene expression

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004109018A1 (fr) 2003-06-06 2004-12-16 Pacheco Pedro Alvares Ribeiro Portique possedant une precontrainte a reglage automatique
WO2017036565A1 (fr) * 2015-09-04 2017-03-09 Universita' Degli Studi Di Padova Procédé de production de cellules souches somatiques
CN107881174A (zh) * 2017-11-22 2018-04-06 张宝会 一种翻译水平基因表达调控的方法及应用
WO2020032057A1 (fr) * 2018-08-07 2020-02-13 Modalis Therapeutics Corporation Nouvel activateur de transcription
RU2800921C2 (ru) * 2018-08-07 2023-08-01 Модалис Терапьютикс Корпорейшн Новый активатор транскрипции

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