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WO1999063074A2 - Methode permettant de modifier le niveau d'expression relatif d'un quelconque gene; procedes et produits connexes - Google Patents

Methode permettant de modifier le niveau d'expression relatif d'un quelconque gene; procedes et produits connexes Download PDF

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Publication number
WO1999063074A2
WO1999063074A2 PCT/US1999/012515 US9912515W WO9963074A2 WO 1999063074 A2 WO1999063074 A2 WO 1999063074A2 US 9912515 W US9912515 W US 9912515W WO 9963074 A2 WO9963074 A2 WO 9963074A2
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Prior art keywords
duplex
sequence
polynucleotide
flanking
heterologous
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PCT/US1999/012515
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English (en)
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WO1999063074A3 (fr
Inventor
Michael J. Lane
Albert S. Benight
Brian D. Faldasz
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Tm Technologies, Inc.
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Publication of WO1999063074A2 publication Critical patent/WO1999063074A2/fr
Publication of WO1999063074A3 publication Critical patent/WO1999063074A3/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
    • C12N15/67—General methods for enhancing the expression

Definitions

  • a means to improve cellular expression of a given biopharmaceutical or to reduce and perhaps inhibit entirely the expression of a repressor polypeptide that interferes with the expression of the desired protein pharmaceutical without altering process validated manufacturing and product purification steps is desirable.
  • the present inventors have previously found that the relative binding of a DNA binding ligand to a site on a duplex DNA is affected by DNA base pair stability of sequences immediately flanking the ligand's binding site (see, e.g., Benight et al. (1995) Adv. Biophys. Chem.. 5: 1-55).
  • this method can be used to alter the expression level of any gene by screening for small molecule pharmacological agents that would bind to polynucleotide sequences adjacent to promoter sites which in turn would either increase or decrease the binding affinity of RNA polymerase and/or other transcription factors for the promoter thereby either increasing or decreasing expression of the gene linked to the promoter (U.S. Patent No. 5,578,444).
  • small molecule pharmacological agents that would bind to polynucleotide sequences adjacent to promoter sites which in turn would either increase or decrease the binding affinity of RNA polymerase and/or other transcription factors for the promoter thereby either increasing or decreasing expression of the gene linked to the promoter.
  • 5,578,444; 5,693,436; 5,716,780; 5,726,014; and 5,738,990 further teach that a ligand binds to its cognate site with "indifference to the nucleotide sequences flanking the screening site.”
  • the binding affinity of a ligand for its binding site is independent of the nucleotide composition of the flanking sequences placed adjacent to the ligand binding site.
  • the present inventors have determined a method of improving the expression of a gene from any given gene locus.
  • the gene may be prokaryotic, eukaryotic, viral or any gene of interest.
  • the prior art which describes the utilization of small molecules which bind to polynucleotide sequences adjacent to promoter sequences and in turn alter the binding characteristics of RNA polymerases and/or transcription factors that transcribe genetic information from DNA to RNA (US. Patent No.
  • the invention described herein alters the ligand-binding characteristics of a nucleic acid sequence of any given length without the use of small molecule pharmaceuticals and teaches that the binding affinity of a ligand, such as RNA polymerase and/or transcription factors, for its ligand binding site may be modulated solely by the nucleotide composition of the polynucleotide sequence(s) flanking an adjacent ligand binding site.
  • a ligand such as RNA polymerase and/or transcription factors
  • this invention features an improved recombinant DNA expression vehicle whose polynucleotide promoter sequence (promoter) is flanked by an adjacent heterologous duplex polynucleotide sequence 5' of the promoter having the nucleotide composition 5 ' -AATTAAATAATAAATTA-3 ' ( SEQ ID NO : l ) and/or a heterologous duplex flanking polynucleotide sequence 3' of the promoter having the nucleotide composition
  • S ' -ATATAAATTAAATATAA-S ' SEQ ID NO : 2
  • transcription factors and/or RNA polymerases to bind the promoter with relatively higher binding affinity when compared to the adjacent native duplex polynucleotide flanking sequences adjacent to the promoter, and thus improves the transcription level of the gene of interest that is linked downstream (3') of the promoter.
  • heterologous adjacent duplex flanking sequences will convert weak promoters to strong promoters and convert already strong promoters to even stronger promoters.
  • the promoter sequence utilized may be a pre-existing promoter sequence available in several commercially available expression vectors.
  • duplex polynucleotide flanking sequences adjacent to the promoters in these expression vectors will be replaced by the heterologous duplex polynucleotide flanking sequences on the 5' (SEQ ID NO:l) and/or 3' (SEQ ID NO:2) side of the promoter by methods well known to those skilled in the art.
  • the heterologous duplex flanking sequences adjacent to the promoter sequence will allow transcription factors and/or RNA polymerase to contact the promoter with relatively higher binding affinity as compared to the adjacent native duplex polynucleotide flanking sequences and initiate transcription at a higher frequency compared to the adjacent native duplex polynucleotide flanking sequences.
  • this invention features an improved recombinant DNA expression vehicle whose polynucleotide promoter sequence (promoter) is flanked by an adjacent heterologous duplex polynucleotide sequence 5' of the promoter having the nucleotide composition
  • heterologous adjacent duplex flanking sequences will convert strong promoters to weak promoters and convert already weak promoters to even weaker promoters.
  • the promoter sequence utilized may be a preexisting promoter sequence available in several commercially available expression vectors.
  • the duplex polynucleotide flanking sequences adjacent to the promoters in these expression vectors will be replaced by the heterologous duplex polynucleotide flanking sequences on the 5' (SEQ ID NO:3) and 3' (SEQ ID NO:4) side of the promoter by methods well known to those skilled in the art.
  • heterologous duplex flanking sequences adjacent to the promoter sequence will allow transcription factors and/or RNA polymerase to contact the promoter with relatively lower binding affinity as compared to the adjacent native duplex polynucleotide flanking sequences and initiate transcription at a decreased frequency compared to the adjacent native duplex polynucleotide flanking sequences.
  • the improved DNA expression vehicle has a novel hetrologous duplex polynucleotide promoter sequence specific for a biologically functional DNA of interest.
  • the sequence of the duplex polynucleotide promoter sequence bound by transcription factor(s) and/or RNA polymerase for the gene of interest that is to be expressed can be determined by methods well known to those skilled in the art (e.g., DNase I footprinting or band shifting).
  • duplex polynucleotide promoter sequence itself can then be amplified by the polymerase chain reaction and native duplex polynucleotide flanking sequences 5' and/or 3' adjacent to the promoter can be replaced by a heterologous duplex polynucleotide sequence 5' of the promoter having the nucleotide composition
  • the improved DNA expression vehicle has a novel hetrologous duplex polynucleotide promoter sequence specific for a biologically functional DNA of interest.
  • the sequence of the duplex polynucleotide promoter sequence bound by transcription factor(s) and/or RNA polymerase for the gene of interest that is to be expressed can be determined by methods well known to those skilled in the art (s g., DNase I footprinting or band shifting).
  • duplex polynucleotide promoter sequence itself can then be amplified by the polymerase chain reaction and native duplex polynucleotide flanking sequences 5' and/or 3' adjacent to the promoter can be replaced by a heterologous duplex polynucleotide sequence 5 ' of the promoter having the nucleotide composition
  • this invention features an improved recombinant DNA expression vehicle whose polynucleotide promoter sequence is flanked by an adjacent heterologous duplex flanking polynucleotide sequence 5' of the promoter sequence, wherein the heterologous duplex flanking polynucleotide sequence is at least 80% homologous to the polynucleotide sequence having the nucleotide composition 5 ' -AATTAAATAATAAATTA-3 ' (SEQ ID NO : l ) and/or an adjacent heterologous duplex flanking polynucleotide sequence 3' of the promoter sequence, wherein the heterologous duplex flanking polynucleotide sequence is at least 80% homologous to nucleotide sequence having the nucleotide composition
  • 5'-ATAT AATTAAATATAA-3' (SEQ ID NO: 2) which allow transcription factors and/or RNA polymerases to bind the promoter with relatively higher binding affinity when compared to the native duplex polynucleotide flanking sequences adjacent to the promoter, and thus improves the transcription level of the gene of interest that is linked downstream (3') of the promoter.
  • Such heterologous adjacent duplex flanking sequences will convert weak promoters to strong promoters and convert already strong promoters to even stronger promoters.
  • the promoter sequence utilized may be a pre-existing promoter sequence available in several commercially available expression vectors.
  • duplex polynucleotide flanking sequences adjacent to the promoters in these expression vectors will be replaced by the heterologous duplex polynucleotide flanking sequences on the 5' (SEQ ID NO:l) and 3' (SEQ ID NO:2) side of the promoter by methods well known to those skilled in the art.
  • the heterologous duplex flanking sequences adjacent to the promoter se_quence will allow transcription factors and/or RNA polymerase to contact the promoter with relatively higher binding affinity as compared to the adjacent native duplex polynucleotide flanking sequences and initiate transcription at a higher frequency compared to the adjacent native duplex polynucleotide flanking sequences.
  • this invention features an improved recombinant DNA expression vehicle whose polynucleotide promoter sequence is flanked by an adjacent heterologous duplex flanking polynucleotide sequence 5' of the promoter sequence, wherein the heterologous duplex flanking polynucleotide sequence is at least 80% homologous to the polynucleotide sequence having the nucleotide composition
  • heterologous duplex flanking polynucleotide sequence is at least 80% homologous to nucleotide sequence having the nucleotide composition
  • RNA polymerases which allow transcription factors and/or RNA polymerases to bind the promoter with relatively lower binding affinity when compared to the native duplex polynucleotide flanking sequences adjacent to the promoter, and thus decreases or significantly reduces the transcription level of the gene of interest that is linked downstream (3') of the promoter.
  • Such heterologous adjacent duplex flanking sequences will convert strong promoters to weak promoters and convert already weak promoters to even weaker promoters.
  • the promoter sequence utilized may be a preexisting promoter sequence available in several commercially available expression vectors.
  • the duplex polynucleotide flanking sequences adjacent to the promoters in these expression vectors will be replaced by the heterologous duplex polynucleotide flanking sequences on the 5' (SEQ ID NO:3) and 3' (SEQ ID NO:4) side of the promoter by methods well known to those skilled in the art.
  • the heterologous duplex flanking sequences adjacent to the promoter sequence will allow transcription factors and/or RNA polymerase to contact the promoter with relatively lower binding affinity as compared to the adjacent native duplex polynucleotide flanking sequences and initiate transcription at a decreased frequency compared to the adjacent native duplex polynucleotide flanking sequences.
  • the improved DNA expression vehicle can also have, in addition to the heterologous duplex polynucleotide sequence of nucleotide composition
  • DNA sequence elements flanking the 3' side of a pre-existing promoter or novel heterologous promoter, DNA sequence elements which act in conjunction with the promoter and contact polypeptide molecules which either induce or repress expression of the gene of interest from the promoter.
  • These unique DNA sequence elements allow the promoter to be regulated by one of several factors which may include pH of the growth medium, growth temperature or composition of the growth medium of the unicellular organism transformed by the improved DNA expression vector. Thus, in the presence of repressors or absence of inducers, expression of the biologically functional gene of interest does not occur. Subsequent to induction or derepression of the promoter, expression of the gene of interest is high.
  • the improved recombinant DNA expression vehicle comprises (i) a duplex polynucleotide enhancer sequence whose naturally occurring adjacent duplex polynucleotide flanking sequence or a pair of naturally occurring adjacent duplex polynucleotide flanking sequences may be replaced by a heterologous duplex flanking sequence or a pair of heterologous duplex flanking sequences; or (ii) a duplex polynucleotide proximal - promoter sequence whose naturally occurring adjacent duplex polynucleotide flanking sequence or a pair of naturally occurring adjacent duplex polynucleotide flanking sequences may be replaced by a heterologous duplex polynucleotide flanking sequence or a pair of heterologous duplex polynucleotide flanking sequences; or (iii) a duplex polynucleotide operator sequence whose naturally occurring adjacent duplex polynucleotide flanking sequence or a pair of naturally occurring adjacent duplex poly
  • heterologous duplex flanking polynucleotide sequence 3' of the enhancer, proximal-promoter, operator, long terminal repeat or promoter is comprised of the nucleotide composition
  • duplex polynucleotide enhancer or duplex polynucleotide proximal-promoter sequence may be located at a great distance either 5' or 3' from the transcription start site of the gene whose expression is to be improved.
  • the duplex polynucleotide promoter sequence is located 5' relative to the gene whose expression is to be improved. The resulting improved DNA expression vehicle will increase the expression of the desired gene.
  • the improved recombinant DNA expression vehicle comprises (i) a duplex polynucleotide enhancer sequence whose naturally occurring adjacent duplex polynucleotide flanking sequence or a pair of naturally occurring adjacent duplex polynucleotide flanking sequences may be replaced by a heterologous duplex flanking sequence or a pair of heterologous duplex flanking sequences; or (ii) a duplex polynucleotide proximal promoter sequence whose naturally occurring adjacent duplex polynucleotide flanking sequence or a pair of naturally occurring adjacent duplex polynucleotide flanking sequences may be replaced by a heterologous duplex polynucleotide flanking sequence or a pair of heterologous duplex polynucleotide flanking sequences; or (iii) a duplex polynucleotide operator sequence whose naturally occurring adjacent duplex polynucleotide flanking sequence or a pair of naturally occurring adjacent duplex polynu
  • heterologous duplex flanking polynucleotide sequence 3 ' of the enhancer, proximal-promoter, operator, long terminal repeat or promoter is comprised of the nucleotide composition
  • duplex polynucleotide enhancer or duplex polynucleotide proximal-promoter sequence may be located at a great distance either 5' or 3' from the transcription start site of the gene whose expression is to be improved.
  • the promoter is located 3' relative to the gene whose expression is to be altered and the resulting improved DNA expression vehicle will synthesize anti-sense mRNA of the gene to which the promoter is associated.
  • the anti-sense mRNA will contact and duplex with the endogenous sense mRNA of the corresponding endogenous gene and prevent or significantly reduce the expression of the gene product.
  • the improved DNA expression vehicle is used in a compatible host cell to enhance the expression of a biologically active polypeptide molecule.
  • the host cell is initially transfected with the improved DNA expression vehicle and cells which stably integrate the expression vehicle into their genomes can be further selected by methods well known to those skilled in the art.
  • the stable transformants can then be grown under appropriate nutrient conditions in which the intact biologically functional mRNA is expressed constitutively and produces the biologically functional polypeptide molecule which is further purified to homogeneity.
  • the improved DNA expression vehicle is used in a compatible host cell to significantly reduce or eliminate the expression of a biologically active polypeptide molecule.
  • the host cell is initially transfected with the improved DNA expression vehicle and cells which stably integrate the expression vehicle into their genomes can be further selected by methods well known to those skilled in the art.
  • the stable transformants can then be grown under appropriate nutrient conditions in which the biologically functional gene product is not expressed.
  • the improved DNA expression vehicle is used in a compatible host cell to enhance expression of a biologically active polypeptide in a regulatable manner.
  • duplex polynucleotide promoter sequence and its adjacent duplex polynucleotide flanking sequence or a pair of duplex polynucleotide flanking sequences will be replaced with a duplex polynucleotide sequence comprising a duplex polynucleotide regulatable promoter flanked on the 5' side by a heterologous duplex polynucleotide flanking sequence having the nucleotide composition
  • the improved DNA expression vehicle is used in a compatible host cell to reduce or eliminate the expression of a biologically active polypeptide in a regulatable manner.
  • the pre-existing duplex polynucleotide promoter sequence and its adjacent duplex polynucleotide flanking sequence or a pair of duplex polynucleotide flanking sequences will be replaced with a duplex polynucleotide sequence comprising a duplex polynucleotide regulatable promoter flanked on the 5' side by a heterologous duplex polynucleotide flanking sequence having the nucleotide composition
  • the relevant pre-exisung duplex polynucleotide promoter sequence and adjacent duplex polynucleotide flanking_s.equences can be replaced by a heterologous duplex promoter sequence and adjacent duplex polynucleotide flanking sequence or a pair of duplex polynucleotide flanking sequences specific for a gene of interest whose biologically functional protein product is to be reduced or eliminated.
  • the expression of the biologically functional polypeptide can be either turned-on or turned-off due to the presence of inducible DNA sequence elements.
  • a "polynucleotide” includes multiple nucleotides (i.e., molecules comprising a sugar (e.g., ribose or deoxyribose) linked to a phosphate group and to an exchangeable organic base, which is either a substituted pyrimidine (e.g. cytosine (C), thymidine (T) or uracil (U)) or a substituted purine (e.g. adenine (A) or guanine (G)).
  • cytosine (C), thymidine (T) or uracil (U) a substituted purine
  • A adenine
  • G guanine
  • polynucleotide refers to both polyribonucleotides and polydeoxyribonucleotides. Polynucleotide sequences can be obtained from existing nucleic acid sources (e.g., genomic DNA or cDNA), but can also be synthetic (e.g.,
  • a "ligand” includes transcription factor(s), RNA polymerase(s) or any compound which when bound to a regulatory sequence, promoter, enhancer, or proximal-promoter sequence, operator or long terminal repeat is able to initiate or inhibit transcription.
  • the transcription factors may be of prokaryotic (e.g., sigma factor of E. Coli) or eukaryotic (e.g., general transcription factors (GTFs), AP-1 or SP-1) origin.
  • RNA polymerases may be of prokaryotic (e.g., RNA polymerase of E. Coli), eukaryotic (e.g., RNA polymerase II) or viral (e.g., bacteriophage T7) origin.
  • ligands may include the lambda cl repressor protein.
  • a "flanking sequence” is a polynucleotide sequence located adjacent to a promoter sequence.
  • a flanking sequence or flanking sequences may be 5', 3' or 5' and 3' of the promoter sequence.
  • a “regulatory sequence” or “polynucleotide regulatory sequence” is a polynucleotide sequence which when contacted by a ligand regulates the transcription of a biologically functional gene associated with the regulatory sequence.
  • promoter any DNA sequence which transcription factor(s) and/or RNA polymerase contacts.
  • RNA polymerase initiates transcription at a high frequency at strong promoters and at low frequency at weak promoters.
  • a "regulatable promoter” is one whose transcription activity can be regulated by changes in pH of the growth medium, growth temperature or composition of the growth medium.
  • Enhancers is any regulatory DNA sequence to which a protein or proteins contact, thereby influencing the rate of transcription of a biologically functional gene associated with the enhancer. Contact of the enhancer by the protein or proteins may either stimulate or decrease the rate of transcription of the associated gene. Enhancers may be located at a great distance either 5' or 3' from the transcription start site of the biologically functional gene it controls. Enhancers may also be regulate transcription of its associated gene when placed in either orientation i.e., 5' ⁇ 3' or 3' ⁇ 5' relative to the gene whose transcription it controls.
  • proximal-promoter sequence or "proximal-promoter element” is any regulatory sequence that is located close to (within 200 base pairs of) a promoter and binds a protein or proteins thereby modulating the transcription of the biologically functional gene associated with the promoter.
  • the proximal-promoter sequence can occur 5' or 3 ' of the transcription start site of the biologically functional gene.
  • An “operator” is a short polynucleotide DNA sequence in a bacterial or viral genome which contacts a protein or proteins and regulates transcription of an associated biologically functional gene.
  • a “Long Terminal Repeat” or “LTR” is a regulatory polynucleotide sequence of viral origin (DNA tumor viruses or retroviruses) comprising integration signals for integrating into the host genome, an enhancer, a promoter and a polyadenylation site.
  • Sequence identity or homology refers to the sequence similarity between two polypeptide molecules or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is compared by adenine, then the molecules are homologous or sequence identical at that position.
  • the percent of homology or sequence identity between two sequences is a function of the number of matching or homologous identical positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 of 10, of the positions in two sequences are the same then the two sequences are 60% homologous or have 60% sequence identity.
  • the DNA sequences ATTGCC and TATGGC share 50% homology or sequence identity.
  • a comparison is made when two sequences are aligned to give maximum homology.
  • "loop out regions" e.g., those arising from deletions or insertions in one of the sequences, are counted as mismatches.
  • the comparison of sequences and determination of percent homology between two sequences can be accomplished using a mathematical algorithm.
  • Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Research 25(17):3389-3402.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • XBLAST and NBLAST can be used. See http://www.nchi.nlm.nih.gov.
  • Another preferred non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989).
  • T7 RNA polymerase Initiation of transcription with T7 RNA polymerase is highly specific for the T7 bacteriophage promoter.
  • Cloning vectors have been developed e.g., pBluescript SK (+/-), which direct transcription from the T7 promoter through polylinker cloning sites.
  • Expression vectors containing the T7 promoter allow in vitro synthesis of defined RNA transcripts and in conjunction with the T7 RNA polymerase gene in vivo synthesis of defined RNA transcripts from a cloned DNA sequence. Under optimal conditions, greater than 700 moles of T7 RNA transcript can be synthesized per mole of DNA template (Noren, C. J. et al, 1990).
  • RNA produced using the T7 RNA polymerase is biologically active as mRNA (Krieg, P. A. and Melton, D. A., 1984) and can be accurately spliced (Green, M. R. et al., 1983).
  • the intent of the present invention is to further increase the synthesis of defined RNA transcripts.
  • EXAMPLE 1 The T7 promoter consensus sequence flanked by a heterologous adjacent polynucleotide sequence or a pair of heterologous adjacent polynucleotide sequences to increase transcription from the promoter
  • the present inventors have previously found that the relative binding of a DNA binding ligand to a site on a duplex DNA is affected by DNA base pair stability of sequences immediately flanking the binding site of the ligand (Benight et al., (1995) Adv.Biophys.Chem. 5:1-55).
  • the polynucleotide sequences immediately flanking the T7 promoter have been replaced by heterologous polynucleotide flanking sequences that increase the binding affinity of T7 RNA polymerase to the T7 promoter polynucleotide sequence by standard molecular cloning techniques well known to those skilled in the art.
  • 5 ' -TAATACGACTCACTATAGGGAGA-3 ' ( SEQ ID NO : 5 ) is flanked on the left (5' of the T7 promoter sequence) by the polynucleotide sequence: 5 ' -TTGTAAAACGACGGCCAGTGAATTG-3 ' ( SEQ ID NO : 6) and on the right (3' of the T7 promoter sequence) by the polynucleotide sequence: 5 ' -CGAATTGGGTACCGGGCCCCCCCTCG-3 ' ( SEQ ID NO : 7 )
  • flanking sequences described above are excised by standard molecular cloning techniques and replaced by heterologous flanking polynucleotide sequences that confer a higher binding affinity for the T7 RNA polymerase as compared to the pre-existing sequences flanking the T7 promoter.
  • the resulting stronger T7 promoter has a higher relative frequency of transcription initiation as compared to the T7 promoter flanked by the preexisting polynucleotide sequences in the commercially available vector.
  • a method of obtaining the optimum sequence composition of the heterologous polynucleotide flanking sequence has been described by the present inventors in US Provisional Application No. 60/068,616. The present inventors find that the following heterologous polyoucleotide sequence located 5' of the T7 promoter:
  • 5 ' -ATATAAATTAAATATAA-3 * confers a higher relative binding affinity for T7 RNA polymerase to the T7 promoter and thus increases the frequency of transcription initiation from the T7 promoter as compared to the original pBluescript SK (+/-) vector.
  • EXAMPLE 2 The T7 promoter consensus sequence flanked by a heterologous adjacent polynucleotide sequence or a pair of heterologous adjacent polynucleotide sequences to decrease transcription from the promoter
  • the output of mRNA transcript driven by the T7 promoter in pBluescript SK(+/-) can also be decreased.
  • sequences immediately flanking the T7 promoter have been replaced by heterologous polynucleotide flanking sequences that decrease the binding affinity of T7 RNA polymerase to the T7 promoter polynucleotide sequence by standard molecular cloning techniques well known to those skilled in the art.
  • flanking sequences described above are excised by standard molecular cloning techniques and replaced by heterologous flanking polyoucleotide sequences that confer a lower binding affinity for the T7 RNA polymerase as compared to the pre-existing sequences flanking the T7 promoter.
  • the resulting weak T7 promoter has a lower relative frequency of transcription initiation as compared to the T7 promoter flanked by the pre-existing polynucleotide sequences in the commercially available vector.
  • a method of obtaining the optimum sequence composition of the heterologous polynucleotide flanking sequence has been described by the present inventors in US Provisional Application No. 60/068,616. The present inventors find that the following heterologous polynucleotide sequence located 5' of the T7 promoter:
  • the scope of this invention is not limited to promoter sequences, but can be used to modify the flanking polynucleotide sequence or a pair of flanking polynucleotide sequences of any regulatory DNA sequence that alters the relative frequency of transcription initiation as compared to the preexisting flanking polynucleotide sequence or a pair of flanking polynucleotide sequences.
  • Enhancers and proximal-promoter sequences are examples of such sequences. Replacing the native flanking polynucleotide sequence or a pair of flanking polynucleotide sequences adjacent to an enhancer can alter the frequency of transcription initiation of the promoter that the enhancer regulates.
  • telomere sequences that regulate the expression of a biologically functional DNA cloned into the vector.
  • pcDNA 3.1(+/-) from Invitrogen® is an example of such an expression vector.
  • the expression of the desired cloned gene can be further improved by replacing the existing polynucleotide flanking sequences with heterologous flanking sequences which confer a higher binding affinity of the promoter-enhancer for transcription factor(s) and/or RNA polymerase, thereby increasing the frequency of transcription initiation and in turn increasing cellular output of the desired product.
  • the ability to increase the cellular output of a biopharmaceutical by simply altering the polynucleotide sequences flanking the promoter of the desired biologically functional gene is of great economic value.
  • the ability to alter the expression of a gene simply by altering the polynucleotide sequences flanking its promoter and/or enhancer also offers great promise in the treatment of human pathological conditions.
  • the present invention can be utilized to increase the expression of a gene whose protein product can be increased to treat a disease in vivo.
  • the STATs Signal Transducers and Activators of Transcription
  • the STATs are a family of latent cytoplasmic proteins that are activated to participate in gene control when cells encounter various extracellular signals.
  • Apoptosis is initiated by activation of a cascade of enzymes that cleave cellular proteins, resulting in the efficient termination of the cell.
  • STATs activate genes containing GAS (gamma-activated sequence) elements.
  • the present invention can be utilized to replace the polynucleotide flanking sequence or a pair of polynucleotide flanking sequences adjacent to the GAS element with a heterologous polynucleotide flanking sequence or a pair of heterologous polynucleotide flanking sequences which confer a relatively higher binding affinity for the STATs as compared to the naturally occurring flanking sequences and thereby increase the expression of genes that which will induce premature death of unwanted cells or cells that have lost control of their own growth.
  • this invention can also be utilized to reduce and perhaps even entirely inhibit the expression of undesirable genes whose protein products can lead to the manifestation of pathological conditions.
  • the naturally occurring adjacent flanking sequence or a pair of naturally occurring adjacent flanking sequences can be excised and replaced by a heterologous flanking polynucleotide sequence or a pair of heterologous polynucleotide flanking 'sequences which confers a relatively lower binding affinity of the transcription factor(s) and/or RNA polymerase for the promoter as compared to the naturally occurring adjacent polynucleotide flanking sequence or a pair of adjacent polynucleotide flanking sequences.
  • the resulting promoter will have a lower relative frequency of transcription initiation as compared to the promoter flanked by the original polynucleotide sequences in the genome and thus reduce or perhaps entirely inhibit the expression of the unwanted gene product.

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Abstract

La présente invention concerne une méthode permettant d'améliorer le niveau d'expression relatif d'un eukaryote, d'un prokaryiote, d'un virus ou de tout autre gène d'intérêt dans des lignées cellulaires eukaryiotes ou prokaryiotes, ainsi que des procédés et produits connexes.
PCT/US1999/012515 1998-06-04 1999-06-04 Methode permettant de modifier le niveau d'expression relatif d'un quelconque gene; procedes et produits connexes WO1999063074A2 (fr)

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US8794598P 1998-06-04 1998-06-04
US60/087,945 1998-06-04
US32324499A 1999-06-03 1999-06-03
US09/323,244 1999-06-03

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CN109750039A (zh) * 2019-01-21 2019-05-14 北京大学 一种植物碰触响应启动子及其应用

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AU691505B2 (en) * 1993-06-17 1998-05-21 Research Foundation Of The State University Of New York, The Thermodynamics, design, and use of nucleic acid sequences
JP2001523952A (ja) * 1997-02-24 2001-11-27 ティーエム テクノロジーズ，インク． アンチセンス・オリゴヌクレオチドを選択するプロセス
AU2011499A (en) * 1997-12-23 1999-07-12 Tm Technologies, Inc. Method of selecting flanking sequences which convey relative binding affinities to a ligand binding site
WO1999042621A2 (fr) * 1998-02-21 1999-08-26 Tm Technologies, Inc. Proprietes thermodynamiques d'acides nucleiques

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