The protected inventions

The group of patents entitled “Tight control of gene expression in eucaryotic cells by tetracycline-responsive promoters” are all directed to sequence encoding a tTA fusion proteincomprising a prokaryotic TetR and a transcriptional activator domain. The claimed tTA binds to tetO sequences in the absence of tetracycline activating the transcription of a gene linked to a tTA responsive promoter.

The US patent US 5464758 claims, in addition to the tTA-encoding sequence:

• a eukaryotic cell transfected with a tTA-encoding sequence and a sequence containing a minimal promoter with tetO sequences linked to a gene of interest; and
• kits containing
  • the tTA-encoding sequence and a second polynucleotide sequence linked to a minimal promoter having at least a tetO sequence; and
  • a eukaryotic cell transfected with the polynucleotide coding for the tTA fusion protein.

The definition of eukaryotic cells provided by the inventors includes yeast, plant cells, insects, mammalian and human cells.

The claims don’t limit the types of regulatory sequences provided with the tTA-encoding sequence.

The patent specification only describes constructs that are constitutively expressed and so although at least some of the claims of this patent dominate those of the Yale patent (US 5851796) discussed above, this patent does not defeat the novelty of the claims of the Yale patent.

The kit claims notably have the same peculiarities of those of the Yale patent, e.g., requiring:

•  at least two container means each containing the a different one of the two molecules (and in one of the kit claims, the tTA-encoding molecule is already in a cell);
• the means have to be in close confinement in the kit.

This claim format may have been copied in the Yale patent from the BASF patent which it referenced by Application No. in the specification. It’s of note that the same law firm listed on the front page of the BASF patent is also listed on the front page of the Yale patent.

BASF’s United States patent US 5650298 claims in addition:

• an isolated DNA sequence coding for a tTA that is recombined with a target DNA molecule. The tTA-coding gene is flanked at each end by DNA sequences that allow homologous recombination with the target sequence.
• In additional claims, these sequences are:
  • a 5′ flanking regulatory region of a gene of interest, and
  • a portion of a gene of interest linked to a tTA-responsive promoter.

In this system, the expression of the tTA is controlled by the promoter of the gene of interest and the target gene is controlled by the tTA-responsive promoter.

Host cells (including mouse stem cells, and human cells of unrestricted type) containing the mentioned sequences, methods for producing the host cells and methods for producing gene products using such sequences are also claimed.

The Canadian patent CA 2165162 claims in addition to the polynucleotide molecules of the United States patent US 5650298:

• the use of tetracycline or a tetracycline analogue for the inhibition of the transcription of a gene of interest linked to a tTA-responsive promoter in a transgenic animal; and
• a method for producing a transgenic animal (non-human) by introducing a tTA coding gene into a fertilized oocyte and implanting the oocyte in a foster mother allowing the oocyte to develop into a transgenic animal.

The Australian patent AU 684524 B2 claims the same type of tTA fusion protein that recombines in a particular location in the host cell as the Canadian patent and the United States patent US 5650298. In addition, the Australian patent claims a transgenic animal (non-human) having the tTA fusion protein integrated at a predetermined location in a chromosome of the animal cells.

The United States patent US 5589362 is directed to a sequence comprising a mutated version of a TetR protein having at least one amino acid mutation, which has the ability to bind a certain class (B) of tetO sequences having a nucleotide substitution at position +4 or +6. The mutated TetR is linked to a polypeptide that regulates transcription in eukaryotic cells. A method for regulating transcription of a class B tetO-linked gene in a cell by introducing into the cell the fusion protein described above.

The fusion proteins, having a first and second polypeptide, claimed in the United States patents US 5654168 and US 5789156 are the opposite from each other. In the first patent, the fusion protein works as a transcriptional activator, whereas in the second, the fusion protein works as a transcriptional inhibitor. The differences between the fusion proteins are as follows:

The United States patent US 5654168 also claims a kit carrying in separate containers:

• a tTA-encoding gene as described above; and
• a nucleotide sequence with a cloning site linked to a tetO sequence of first class type.

The United States patent US 5789156 also claims a kit carrying in separate containers:

• a fusion protein as described above or a eukaryotic cell line into which the fusion protein has been introduced; and
• a nucleotide sequence with a cloning site linked to at least a tetO sequence.

The independent claims of the patents entitled “Methods for regulating gene expression” are methods for regulating the expression of a tetO-linked gene in a cell. The expression is regulated by the concentration of tetracycline administered to the cell. The differences between the independent claims of the three United States patents are:

• US 5814618
  • in one method the fusion protein encoded by the gene introduced in the cell contains a TetR and a second polypeptide that inhibits transcription in the cell; and
  • in a second method two different nucleotide molecules are introduced in a cell:
    • a fusion protein-encoding gene as mentioned above and
    • a nucleotide sequence linked to at least a tetO sequence.
• US 5888981
  the fusion protein used in the methods claimed is a tetracycline-controllable transactivator (tTA), which activates the transcription in eukaryotic cells.
• US 6004941
  the TetR of the fusion protein binds to the tetO sequences in the presence of tetracycline, activating transcription of the gene linked to the tetO sequences.In addition, there are two product claims directed to recombinant vectors containing:
  • in one case, two different nucleotide sequences comprising each a cloning site linked to the same tetO sequence for bidirectional regulation of the transcription; and
  • in another case, each of the two cloning sites is linked to different class of tetO sequence for independent regulation of the transcription.

The United States patents US 5859310, US 5866755, US 5912411 and US 5922927 are all directed to transgenic mice containing a tetracycline-responsive transcriptional regulator. The transgenic mice of the inventions have integrated in their genome:

• a transgene encoding a fusion protein, and
• a gene of interest linked to a tetO sequence.

The transcription of the gene of interest is either activated or inhibited by the binding of the fusion protein to the tetO sequences. The fusion protein formed by two different polynucleotides differs between the patents as follows:

The United States patent US 5922927 claims methods for producing a transgenic mouse with a transgene coding for a fusion protein as described in the table. Some of the methods involve the introduction of a tTA-encoding sequence in a fertilized oocyte and its implantation in a foster mother, allowing the development of a transgenic mouse. In other methods, the tTA-encoding gene is introduced in embryonic stem cells of a mouse. For homologous recombination between the tTA-encoding transgene and the gene of interest in the cells of a mouse, the DNA molecule carrying the transgene comprises:

• a 5′ regulatory region of the gene of interest linked to,
• a tTA-encoding sequence, and
• a tTA-responsive promoter linked to
• at least a portion of the gene of interest of sufficient length to mediate homologous recombination.

The fusion protein of the United States patent US 6136954 activates transcription in the presence of tetracycline. A portion of the fusion protein binds to the tetO.  It’s not limited to the tetR and the patent describes that mutated tetR proteins are part of the invention.The second polypeptide of the fusion protein is a transcriptional activator.  In the broadest claims of the patent, the activator is also not limited as to structure or amino acid sequence so long as it functions as an activator (though the level of activation required is not defined).

Conversely, the second polypeptide of the fusion protein claimed in the United States patent US 6271348 is a transcriptional inhibitor and therefore, the protein as a whole inhibitstranscription in eukaryotic cells. The inhibitor portion also has no structural limitations in the broadest claims.  The term “inhibition” is defined in the patent as “a diminution in the level or amount of transcription of a target gene compared to the level or amount of transcription prior to regulation by the transcriptional inhibitor protein.”  The patent describes that transcriptional inhibition may be partial or complete.

The United States patents entitled “Transgenic organisms having tet racycline-regulated transcriptional regulatory systems” are directed to transgenic plants containing:

• a tetO-linked gene of interest and
• a transgene comprising a fusion protein having a first and a second polypeptide that either activates or inhibits the transcription of the gene linked to the tetO sequence as follows:

The claims of the granted Australian patent  AU 746850 B2 are generally drawn to:

• An isolated DNA sequence coding for a fusion protein comprising:
  • a first polypeptide that binds to tetO in the presence of tetracycline, and operatively linked to
  • a transcriptional activator for eukaryotic cells.
• The above fusion protein which activates transcription.
• An isolated nucleic acid sequence coding for a fusion protein where the second polypeptide inactivates transcription.
• The above fusion protein which inhibits transcription.
• A host cell and an organism (except humans) comprising both types of fusion proteins (activating and inhibiting transcription proteins) and a sequence of interest to be transcribed linked to a tetO sequence.
• A recombinant vector for bidirectional transcription of genes of interest. There are cloning sites at each end of a tetO sequence for the introduction of the sequences to be transcribed.
• A recombinant vector for independent regulation of transcription of two genes of interest where the tetO sequences linked to the genes are of a different class.
• A kit comprising in separate containers
  a fusion protein that activates transcription in the presence of tetracycline and
  a DNA sequence comprising a cloning site for inserting the gene of interest linked to a tetO sequence.

The first polypeptide of the fusion proteins is not specified in the independent claims.

The only independent claim as filed of the EP application 1092771 recites a DNA sequence encoding a fusion protein that inhibits transcription in eukaryotic cells. The first polypeptide of the fusion protein is defined as a TetR.