Syngenta patent families on positive selection

Syngenta Participations AG owns two patent families (here named as A and B) on positive selection systems.  The assignments were previously to Novartis AG, one of the precursor companies that formed Syngenta, which had acquired certain patents from Danisco.

The patent family A, analysed in depth below, contains broad patents directed to a general method of selecting genetically transformed cells from a population of transformed and non-transformed cells by introducing a nucleotide sequence into plant cell so that the transformed cells have a competitive advantage in utilizing a compound; if covered by the claims, the introduced nucleotide sequence is not a marker gene for toxin, antibiotic or herbicide resistance, which are found in the “prior art”.

The patent family B, analysed on a following page, is much less broad.  It is directed more specifically to a method of selecting genetically transformed cells from a population of transformed and non-transformed cells by transforming cells with a gene coding for an enzyme involved in mannose or xylose metabolism and selecting on a medium supplied with a compound that only the transformed cells are able to utilize. Such enzymes include xyloisomerases and phosphomanno-isomerases (such as mannose-6-phosphate isomerase and mannose-1-phosphate isomerase), phosphomanno mutase, mannose epimerases (those which convert carbohydrates to mannose or mannose to carbohydrates such as glucose or galactose); phosphatases (such as mannose-6-phosphatase and mannose-1-phosphatase), and permeases which are involved in the transport of mannose, or a derivative, or a precursor thereof into the cell.

Syngenta patent family A

This patent family has patents on positive selection granted in the United States, Europe, Canada, Australia and some other jurisdictions, as indicated in the patent information table at the bottom of this page.

Following Novartis’ application for the European patent (EP 601092 B1) (and the US patent 5994629), oppositions were lodged in Europe by CAMBIA, BASF AG and Unilever PLC (opposition is not a process currently available in the United States).  Information that was presented in the European opposition for invalidating some of the claims in EP 601092 is provided here relative to the claims of US 5994629 (this example of validity analysis on patent claims was provided for CAMBIA by Foley and Lardner).

Validity analysis of certain claims of US 5994629

I. The Prosecution History of the Patent Family

A. Priority Claims and Patent Family Information

The US 5994629 patent is a continuation-in-part of Application No. 08/505, 302, filed on 3 October 1995, now U.S. Patent US 5767378. The US patents 5994629 and 5767378 each claim priority to GB 9304200 filed on 2 March 1993.

The US 5994629 patent is also a continuation-in-part of Application No. 08/378, 996, filed on 27 January 1995, now abandoned.  Application No. 08/378, 996, claims priority to application No. 08/196, 152, now abandoned, which was originally filed as application PCTIDK92/00252 on 27 August 1992. The US 5994629 patent, application No. 08/378, 996, application No. 08/196, 152 and PCT /DK92/00252 all claim priority to DK 1522/91, filed on 28 August 1991.  For the relationships between the applications, see diagram below.

B. Amendment History of the Claims of the US 5994629 Patent

According to the prosecution history of the US 5994629 patent, the applicants amended each of claims 1 and 7.  During prosecution in the USPTO, the applicants deleted “induces a positive effect” and “and” from claim 1.   Also in claim 1, the applicants added the limitations”…expression or transcription of…and…that only the transformed cells are able to utilize…”.   In claim 7, the applicants deleted “a positive effect induced by” and added “…that only the transformed cells are able to utilize…”.  In support of the amendments, the applicants argued, “claims 1, 7 and 26 are amended to clarify that the competitive advantage of transformed cells is due to the expression or transcription of the co-introduced nucleotide sequence.  Claims 1 and 7 are further amended to clarify that only transformed cells are able to utilize the ‘supplied compound’ by expression or transcription of the co-introduced nucleotide sequence and therefore have a competitive advantage.”

The amendments to the claims are significant because the deletion of the limitation “induces a positive effect” broadens the scope of the claims such that the claims are open to negative effects.  However, the addition of the limitation “that only the transformed cells are able to utilize” narrows the scope of the claims by excluding compounds that may be used to a lesser extent by non-transformed cells, as compared to transformed cells.

C. References cited

During the prosecution of the US 5994629 patent, the applicants’ attorney cited 3 references. The examiner cited no references and included a form PTO-892 with “NONE” written across its face.  None of the references from the parent applications of the US 5994629 patent or from the International Search Report or International Preliminary Examination Report of PCT /DK92/00252 were cited by either the examiner or the applicants in the prosecution of the US 5994629 patent.

II. Validity Analysis

A. Relevant Documents

The following prior art references are relevant to the validity analysis:

Doc1:  Jefferson, R. A. (1990), Gene Manipulation and Plant Improvement II, “New approaches for agricultural molecular biology: from single cells to field analysis,” pp. 365-400., which qualifies under the provisions of 35 U.S.C. 102(b) as prior art because it described the invention claimed in the US 5994629 patent in a printed publication in 1990, more than one year prior to 27 August 1992, the earliest effective U.S. filing date for the US 5994629 patent.

Doc2:  Budar et al. (1986) “Introduction and expression of the octopine T-DNA oncogenes in tobacco plants and their progeny,” Plant Science 46:195-206, which qualifies as prior art against the US 5994629 patent under the provisions of 35 U.S.C. 102(b) because it was published in 1986, more than one year prior to 27 August 1992.

Doc3: Jefferson R. A. (filed: 8 December 1989; earliest priority: 11 November 86) U.S. Patent 5268463 issued 7 December 1993, which qualifies as prior art against the US 5994629 patent under the provisions of 35 U.S.C. 102(e), because although issued after the earliest priority date for the US 5994629 patent, it was filed in the U.S. prior to 28 August 1991.

Doc4:  Sreekrishna et al. (filed 23 July 1986; earliest priority: February 1984) U.S. Patent No. 4857467 issued: 15 August 1989, which qualifies as prior art against the US 5994629patent under the provisions of 35 U.S.C. 102(b) because it was published in 1989, more than one year prior to 27 August 1992.

Doc5:  Liijestroem et al. (14 October 1987) EPO 0241044 published application, which qualifies as prior art against the US 5994629 patent under the provisions of 35 U.S.C. 102(b) because it was published in 1987, more than one year prior to 27 August 1992.

Doc6:  von Schaewen et al. (1990) “Expression of a yeast-derived invertase in the cell wall of tobacco and Arabidopsis plants leads to accumulation of carbohydrate and inhibition of photo synthesis and strongly influences growth and phenotype of transgenic tobacco plants,” EMBO Journal 9:3033-3044, which qualifies as prior art under the provisions of 35 U.S.C. 102(b) because it described the invention in a printed publication in 1990, more than one year prior to 27 August 1992.

B. Summary of Analyzed Claims

For convenience, claim 1 of US 5994629 was broken into the following individual elements (the reason this is useful will be clear in the analysis of prior art, Section C below):

[1.1] Genetically transformed plant cells comprising

[1.2] a desired nucleotide sequence and

[1.3] a co-introduced nucleotide sequence

[1.4] wherein expression or transcription of the co-introduced nucleotide sequence in the transformed cells gives said transformed cells a competitive advantage

[1.5] when a population of cells including the transformed and the non-transformed cells is supplied with a compound

[1.6] that only the transformed cells are able to utilize, and

[1.7] the desired nucleotide sequence codes for a gene other than a toxin, antibiotic or herbicide resistance gene.

Likewise, claim 7 was broken into the following individual elements:

[7.1] A method of selecting genetically transformed cells from a population of cells comprising the steps of:

[7.2] a) introducing into the genome of a plant cell a desired nucleotide sequence and

[7.3] a co-introduced nucleotide sequence

[7.4] wherein said desired nucleotide sequence or co-introduced nucleotide sequence codes for a sequence other than a toxin, antibiotic or herbicide resistance gene;

[7.5] b) obtaining transformed cells;

[7.6] c) supplying to the population of cells a compound

[7.7] that only transformed cells are able to utilitize

[7.8] wherein said transformed cells have a competitive advantage over non-transformed cells due to the expression or transcription of the desired nucleotide sequence or co-introduced nucleotide sequence in the       presence of the compound; and

[7.9] d) selecting said transformed cells from the population of cells.

C.  Validity Analysis of Claim 1 under 35 U.S.C. 102

1. Claim 1 of the US 5994629 patent fails to meet the requirements of 35 U.S.C. 102(b) because it is anticipated by the Jefferson article (Doc1)

All of the elements of claim 1 are taught by the Jefferson article.

With regard to element 1.1, the Jefferson article discloses genetically transformed plant cells. Specifically, the Jefferson article discloses transformed tobacco plants. (p. 396, 1st full paragraph, lines 10-12).

With regard to elements 1.2 and 1.3, the Jefferson article discloses tobacco plants transformed with a CaMV 35S – GUS fusion. (p. 396, 1st full paragraph, lines 10-12) and further describes a gene fusion as “DNA constructions in which DNA sequences from two (or more) genes are combined.” (p. 369, 2nd full paragraph, lines 1-4).

With regard to element 1.4, the Jefferson article discloses an experiment wherein the transformed cells have a competitive advantage over non-transformed cells. Specifically, the transformed cells which incorporate the GUS fusion can use tryptophyl-A-glucuronide as an auxin source, and the non-transformed cells cannot (p. 396, 1st full paragraph).  Since the transformed cells can use at least one more compound as an auxin source as compared to the non-transformed cells, the transformed cells have a competitive advantage over the non¬transformed cells.

With regard to element 1.5, the Jefferson article describes an experiment wherein non-transformed cells and transformed cells are supplied with tryptophyl-A-glucuronide (p. 396, 1st full paragraph).

With regard to element 1.6, the Jefferson article, in the same experiment states that tryptophyl-A-glucuronide “shows no auxin activity … when assayed using untransformed cells,” and indicates that the transformed cells “remained green and healthy,” thus indicating that the transformed cells used the tryptophyl-A-glucuronide as an auxin source (p. 396, 1st full paragraph).

With regard to element 1.7, the Jefferson article discloses that the GUS gene “catalyzes hydrolysis of a very wide variety of A-glucuronides” and further discloses that “gene fusions are DNA constructions in which DNA sequences from two (or more) genes are combined such that the coding sequences of one gene (the responder),” e.g. here the GUS gene, “are transcribed and/or translated under the direction of another gene(s) (the controller),” i.e. here the CaMV 35S sequence (p. 371, 1st full paragraph, lines 5-6).   Thus, “the desired nucleotide sequence” directs transcription or translation of the GUS gene and does not code for a “toxin, antibiotic or herbicide resistance gene.”

2. Claim 1 of the US 5994629 patent fails to meet the requirements of 35 U.S.C. 102(b) because it is anticipated by the Budar et al. article (Doc2)

All of the elements of claim 1 are taught by the Budar et al. article.

With regard to element 1.1, the Budaret al. article discloses the introduction of genes into tobacco cells by transformation and “normal transformed plants.” (Abstract).

With regard to elements 1.2 and 1.3, the Budar et al. article discloses that “genes 1, 2, and 4. . . were cloned and introduced into tobacco cells by…leaf disk transformation.” (Abstract)

With regard to element 1.4, the Budar et al. article discloses that the “product of genes 1 and 2 are involved in the production of the auxin …” (p. 195, 2nd column, 1st full paragraph, lines 6-14).

With regard to elements 1.5 and 1.6, the Budar et al. article discloses that “our data show that genes 1 and 2 together … can be used for positive and negative selections. One can select for the expression of gene 2 in plant cells if α-naphthalene acetamide is provided in the medium instead of an active auxin,” (p. 205, 1st column, 4th full paragraph, lines 1-7).

Unless a cell population contained both transformed cells and non-transformed cells, there would be no need for selection. Further, if the non-transformed cells were able to use the α-naphthalene acetamide, there would be no way to select the transformed cells which, as noted, are able to use the α-naphthalene acetamide. Therefore, the disclosure of the Budar article sets forth providing a population of transformed and non-transformed cells with a compound that only the transformed cells are able to utilize.

With regard to element 1.7, the Budar et al. article discloses that “the protein encoded by gene 1 catalyzes the formation of indole-3-acetamide which is converted to IAA by the product of gene 2” (p. 195, 2nd col., 1st full paragraph, lines 6-14).  IAA, indole acetic acid, is an auxin (p. 195, 2nd full paragraph, lines 6-14).  “The product of gene 2 also catalyzes the formation of naphthalene acetic acid (NAA) when alpha-naphthalene acetamide is provided” (p. 195, 2nd col., 1st full paragraph, lines 6-14).

3. Claim 1 of the US 5994629 patent fails to meet the requirements of 35 U.S.C. 102(b) because it is anticipated by U.S. Patent 5268463 (Doc3)

All of the elements of claim 1 are taught by U.S. Patent 5268463.

With regard to element 1.1, U.S. Patent 5268463 discloses “plants transformed with a highly expressed CaMV 35S/GUS gene fusion.” (col. 56, lines 2-3).

With regard to elements 1.2 and 1.3, U.S. Patent 5268463 discloses “a gene fusion comprising a GUS encoding nucleic acid under the control of a promoter/enhancer element, X, could be used to generate a transgenic plant. Tissue-specific activity of promoter X would be detectable by the observation that GUS activity was expressed in some plant tissue, but not others” (col. 20, lines 4-10).  Specifically U.S. Patent 5268463 discloses a CaMV 35S/GUS gene fusion, i.e. a desired nucleotide sequence and a co-introduced nucleotide sequence.

With regard to element 1.4, U.S. Patent 5268463 discloses an experiment in which “in the absence of auxin, leaf discs from control plants and CaMV 35S/GUS-transformed ‘GUS plants’ became chlorotic and died over a 7 week period (Fig. 18) … on media in which tryptophyl glucuronide was the sole auxin source, only those leaves which expressed GUS remained green and healthy, presumably because they were able to cleave active auxin from tryptophyl glucuronide” (col. 56, lines 15-25).

With regard to element 1.5, U.S. Patent 5268463 discloses that “leaf discs from nontransformed and CaMV 35S/GUS transformed plants were exposed to media containing cytokinin and (i) no auxin, (ii) 1 μM tryptophyl-glucuronide, or (iv) 10 μM tryptophyl glucuronide.” (co1. 56, lines 1-7). Thus, transformed and non-transformed cells were supplied with a compound.

With regard to element 1.6, U.S. Patent 5268463 discloses, in describing the outcome of the above mentioned experiment (iv) that “only those leaves that expressed GUS remained green and healthy, presumably because they were able to cleave active auxin from tryptophyl glucuronide” (coI. 56, lines 22-25).

With regard to element 1.7, U.S. Patent 5268463 discloses “a gene fusion comprising a GUS encoding nucleic acid under the control of a promoter/enhancer element, X, could be used to generate a transgenic plant.  Tissue-specific activity of promoter X would be detectable by the observation that GUS activity was expressed in some plant tissue, but not others” (col. 20, lines 4-10).  Specifically U.S. Patent 5268463 discloses a CaMV 35S/GUS gene fusion, e.g. a desired nucleotide sequence and a co-introduced nucleotide sequence. (col. 56, lines 1-4).  The CaMV 35S serves here as a promoter, (col 49, lines 6-7).

D. Validity Analysis of Claim 7 under 35 U.S.C. 102

1.  Claim 7 of the US 5994629 patent fails to meet the requirements of 35 U.S.C. 102(b) because it is anticipated by the Jefferson article (Doc1)

All of the elements of claim 7 are taught by the Jefferson article.

With regard to element 7.1, the Jefferson article describes an experiment wherein non-transformed cells and transformed cells are supplied with tryptophyl-A-glucuronide. (p. 396, 1st full paragraph). In the same experiment the Jefferson article discloses that tryptophyl-A-glucuronide “shows no auxin activity … when assayed using untransformed cells,” and indicates that the transformed cells “remained green and healthy,” thus indicating that the transformed cells used the tryptophyl-A-glucuronide as an auxin source (p. 396, 1st full paragraph).  Since the transformed cells can use at least one more compound as an auxin source as compared to the non-transformed cells, the transformed cells have a competitive advantage over the non-transformed cells (p. 396. 1st full paragraph). The discussion of the experimental results concludes with the statement “other compounds are now being synthesized to achieve both negative and positive effects” (p. 396, 1st full paragraph).  Taken in the context of the discussion of “Fusion Genetics – Positive and Negative Selection for Gene Fusion Action” on p. 394, the term “negative and positive effects” on p. 396 clearly refers to negative and positive selection.

With regard to elements 7.2 and 7.3, the Jefferson article discloses tobacco plants transformed with a CaMV 35S – GUS fusion (p.396, 1st full paragraph, lines 10-12). The Jefferson article further describes a gene fusion as “DNA constructions in which DNA sequences from two (or more) genes are combined” (p. 369, 2nd full paragraph, lines 1-4).

With regard to element 7.4, the Jefferson article discloses that the GUS gene “catalyzes hydrolysis of a very wide variety of glucuronides” (p. 371, 1st full paragraph, lines 5-6). The Jefferson article further discloses that “gene fusions are DNA constructions in which DNA sequences from two (or more) genes are combined such that the coding sequences of one gene (the responder),” i.e. here the GUS gene, “are transcribed and/or translated under the direction of another gene(s) (the controller),” e. g. here the CaMV 358 gene.  Thus, neither the GUS gene nor the CaMV 35S gene code for a “toxin, antibiotic or herbicide resistance gene.”

With regard to element 7.5, the Jefferson article discloses genetically transformed plant cells, specifically transformed tobacco plants (p. 396, 1st full paragraph, lines 10-12).

With regard to element 7.6, the Jefferson article describes an experiment wherein non-transformed cells and transformed cells are supplied with tryptophyl-A-glucuronide (p. 396, 1st full paragraph),

With regard to element 7.7, the Jefferson article, in the same experiment, states that tryptophyl-A-glucuronide “shows no auxin activity…when assayed using untransformed cells,” and indicates that the transformed cells “remained green and healthy,” thus indicating that the transformed cells used the tryptophyl-A-glucuronide as an auxin source (p. 396, 1st full paragraph).

With regard to element 7.8, the Jefferson article discloses an experiment wherein the transformed cells have a competitive advantage over non-transformed cells.  Specifically, the transformed cells which incorporate the GUS fusion can use tryptophyl-A-glucuronide as an auxin source, and the non-transformed cells cannot.  Since the transformed cells can use at least one more compound as an auxin source as compared to the non-transformed cells, the transformed cells have a competitive advantage over the non-transformed cells (p. 396, 1st full paragraph).

With regard to element 7.9, the Jefferson article describes an experiment wherein non-transformed cells and transformed cells are supplied with tryptophyl-A-glucuronide (p. 396, 1st full paragraph).  In the same experiment the Jefferson article discloses that tryptophyl-A-glucuronide “shows no auxin activity … when assayed using untransformed cells,” and indicates that the transformed cells “remained green and healthy,” thus indicating that the transformed cells used the tryptophyl-A-glucuronide as an auxin source (p. 396, 1st full paragraph).  Since the transformed cells can use at least one more compound as an auxin source as compared to the non-transformed cells, the transformed cells have a competitive advantage over the non-transformed cells (p. 396, 1st full paragraph).  The discussion of the experimental results concludes with the statement “other compounds are now being synthesized to achieve both negative and positive effects” (p. 396, 1st full paragraph).  Taken in the context of the discussion of “Fusion Genetics – Positive and Negative Selection for Gene Fusion Action” on p. 394, the term “negative and positive effects” on p. 396 clearly refers to negative and positive selection.

2. Claim 7 of the US 5994629 patent fails to meet the requirements of 35 U.S.C. 102(b) because it is anticipated by the Budar et al. article (Doc2)

All of the elements of claim 7 are taught by the Budar et al. article.

With regard to element 7.1, the Budar et al. article discloses that “genes 1 and 2 together or gene 2 associated with α-naphthalene acetamide can be used for positive and negative selections. One can select for the expression of gene 2 in plant cells if α-naphthalene acetamide is provided in the medium instead of an active auxin.” (p. 205, 1st col., 4th full paragraph, lines 1-7).

With regard to elements 7.2 and 7.3, the Budar et al. article discloses that “genes 1, 2, and 4 … were cloned and introduced into tobacco cells by … leaf disk transformation. ” (Abstract).

With regard to element 7.4, the Budar et al. article discloses that “the protein encoded by gene 1 catalyzes the formation of indole-3-acetamide which is converted to IAA by the product of gene 2.”  IAA, indole acetic acid, is an auxin.  “The product of gene 2 also catalyzes the formation of naphthalene acetic acid (NAA) when α-naphthalene acetamide is provided” (p. 195, 2nd col, 1st full paragraph, lines 6-14).

With regard to element 7.5, the Budar et al. article discloses the introduction of genes into tobacco cells by transformation and “normal transformed plants.” (Abstract).

With regard to elements 7.6 and 7.7, the Budar et al. article discloses that “our data show that genes 1 and 2 together… can be used for positive and negative selections. One can select for the expression of gene 2 in plant cells if α-naphthalene acetamide is provided in the medium instead of an active auxin” (p. 205, 1st column, 4th full paragraph, lines 1-7).  Unless a cell population contained both transformed cells and non-transformed cells, there would be no need for selection.  Further, if the non-transformed cells were able to use the α-naphthalene acetamide, there would be no way to select the transformed cells which, as noted, are able to use the α-naphthalene acetamide.  Therefore, the disclosure of the Budar et al. article sets forth providing a population of transformed and non-transformed cells with a compound that only the transformed cells are able to utilize.

With regard to element 7.8, the Budar et al. article discloses that the “product of genes 1 and 2 are involved in the production of the auxin …” (p. 195, 2nd col, 1st full paragraph, lines 6-14).

With regard to element 7.9, the Budar et al. article discloses that “genes 1 and 2 together or gene 2 associated with α-naphthalene acetamide can be used for positive and negative selections.  One can select for the expression of gene 2 in plant cells if α-naphthalene acetamide is provided in the medium instead of an active auxin” (p. 205, 1st col., 4th full paragraph, lines 1-7).

3. Claim 7 of the US 5994629 patent fails to meet the requirements of 35 U.S.C. 102(b) because it is anticipated by U.S. Patent 5268463 (Doc3)

All of the elements of claim 7 are taught by U.S. Patent 5268463.

With regard to element 7.1, U.S. Patent 5268463 discloses an experiment wherein “on media in which tryptophyl glucuronide was the sole auxin source, only those leaves which expressed GUS remained green and healthy, presumably because they were able to cleave active auxin from tryptophyl glucuronide” (col. 56, lines 20-26). U.S. Patent 5268463further discloses that “to identify plants which express Y, one may identify plants that express GUS, as the expression of both genes is under the control of the same promoter” (col. 20, lines 44-50). Additionally U.S. Patent 5268463 discloses that “GUS gene fusions could be used to report on the expression of a second gene of interest” (col. 20, lines 16-19).

With regard to elements 7.2 and 7.3, U.S. Patent 5268463 discloses a gene fusion comprising a GUS encoding nucleic acid under the control of a promoter/enhancer element X, could be used to generate a transgenic plant.  Tissue-specific activity of promoter X would be detectable by the observation that GUS activity was expressed in some plant tissue, but not others” (col. 20, lines 4-10).  Specifically U.S. Patent 5268463 discloses a CaMV 35S/GUS gene fusion, i.e. a desired nucleotide sequence and a co-introduced nucleotide sequence.

With regard to element 7.4, U.S. Patent 5268463 discloses “a gene fusion comprising a GUS encoding nucleic acid under the control of a promoter/enhancer element X, could be used to generate a transgenic plant. Tissue-specific activity of promoter X would be detectable by the observation that GUS activity was expressed in some plant tissue, but not others” (col. 20, lines 4-10).  Specifically U.S. Patent 5268463 discloses a CaMV 35S/GUS gene fusion, e.g. a desired nucleotide sequence and a co-introduced nucleotide sequence (coI. 56, lines 1-4).  The CaMV 35S sequence serves here as a promoter (col. 49, lines 6-7).

With regard to element 7.5, U.S. Patent 5268463 discloses “plants transformed with a highly expressed CaMV 35S/GUS gene fusion” (col. 56, lines 2-3).

With regard to element 7.6, U.S. Patent 5268463 discloses that “leaf discs from nontransformed and CaMV 35S/GUS transformed plants were exposed to media containing cytokinin and (i) no auxin, (ii) 1 μM tryptophyl-glucuronide, or (iv) 10 μM tryptophyl glucuronide” (col. 56, lines 1-7).  Thus, transformed and non-transformed cells were supplied with a compound.

With regard to element 7.7, U.S. Patent 5268463 discloses, in describing the outcome of the above mentioned experiment (iv) that “only those leaves that expressed GUS remained green and healthy, presumably because they were able to cleave active auxin from tryptophyl glucuronide” (col. 56, lines 22-25).

With regard to element 7.8, U.S. Patent 5268463 discloses an experiment in which “in the absence of auxin, leaf discs from control plants and CaMV 35S/GUS-transformed ‘GUS plants’ became chlorotic and died over a 7 week period (Fig. 18)…on media in which tryptophyl glucuronide was the sole auxin source, only those leaves which expressed GUS remained green and healthy, presumably because they were able to cleave active auxin from tryptophyl glucuronide” (col. 56, lines 15-25).

With regard to element 7.9, U.S. Patent 5268463 discloses an experiment wherein “on media in which tryptophyl glucuronide was the sale auxin source, only those leaves which expressed GUS remained green and healthy, presumably because they were able to cleave active auxin from tryptophyl glucuronide” (col. 56, lines 20-26). U.S. Patent 5268463further discloses that “to identify plants which express Y, one may identify plants that express GUS, as the expression of both genes is under the control of the same promoter” (col. 20, lines 44-50).  Additionally, U.S. Patent 5268463 discloses that “GUS gene fusions could be used to report on the expression of a second gene of interest” (col. 20, lines 16-19).

E.  Analysis of Dependent Claims 12, 21 and 22 under 35 U.S.C. 102

The validity of dependent claims 12, 21 and 22 of the US 5994629 patent is also analyzed here.  Each of these claims depends either directly or indirectly from claim 7 analysed above.

1. Dependent claims 12 and 22 of the US 5994629 patent fail to meet the requirements of 35 U.S.C. 102(b) because they are anticipated by the Jefferson article (Doc1)

All of the elements of claims 12 and 22 are taught by the Jefferson article.

With regard to dependent claim 12, the Jefferson article discloses that “the gus operon consists of the glucuronidase gene itself, encoding GUS (gusA – formerly uidA) … “. (p. 391, 1st full paragraph, lines 1-2).  The Jefferson article further discloses “Table 1. GUS β-Glucuronidase … Encoded by E. coli gusA (formerly uidA)” (p. 373, lines 1-2).

With regard to dependent claim 22, the Jefferson article discloses that “many other compounds are now being synthesized to achieve both positive and negative effects, for instance cyclohexamide-glucuronide, cytokinin glucuronide, etc.” (p. 396, 1st full paragraph, lines 17-20).

2. Dependent claims 12, 21 and 22 of the US 5994629 patent fail to meet the requirements of 35 U.S.C. 102(b) because they are anticipated by U.S. Patent 5268463(Doc3)

All of the elements of claims 12,21 and 22 are taught by U.S. Patent 5268463.

With regard to dependent claim 12, U.S. Patent 5268463 discloses transgenic plants expressing a β-glucuronidase gene fusion (col. 55, lines 60-63). U.S. Patent 5268463 further discloses that “the present invention relates to the β-glucuronidase (GUS) gene fusion…it is based on the surprising discovery that gene fusions comprising β-glucuronidase gene may be effectively expressed in a wide variety of organisms to produce active β-glucuronidase enzyme.” (Abstract).

With regard to dependent claim 21, the Jefferson Patent discloses that “an additional and sometimes very useful technique is to use the specific β-glucuronidase inhibitor saccharolactone (Levvy, G. A., 1952, Biochem. J. 52:464) (Sigma S-0375, saccharic acid 1-4 latone, glucaric acid 1-4 lactone; glucarolactone) to corroborate the GUS-dependence of the fluorescence increase.  This inhibitor will eliminate glucuronidase activity at concentrations less than one millimolar, but the compound is unstable at neutral pH, so that care should be exercised during prolonged assays.  Because of this instability, it is preferable to use saccharolactone at up to 5 mM for assays up to half an hour.  Alternatively, the reaction and the inhibited reaction may preferably be performed at pH 6.0 or below.  GUS activity should not be affected by these conditions and saccharolactone is more stable at acid pH” (col. 31, lines 19-34).

With regard to dependent claim 22, U.S. Patent 5268463 discloses”…glucuronides comprising bioactive molecules can also be used as GUS substrates according to the invention; useful bioactive compounds include, but are not limited to steroid hormones non-steroid hormones and factors, lymphokines, auxins, cytokinins…” (col. 26, lines 40-47).

F.  Conclusion on the validity analysis

Based on the validity analysis, the folowing conclusion can be made:

A well-informed court should conclude that claims 1 and 7 of the US 5994629 patent are invalid because they fail to meet the requirements of 35 U.S.C. 102 in view of the disclosures of any one of the Jefferson article (Doc1), the Budar et al. article (Doc2), and U.S. Patent 5268463 (Doc3).

Furthermore, claims 12 and 22, which depend from to claim 7, are invalid as anticipated by either the Jefferson article or U.S. Patent 5268463.  Claim 21 is invalid as anticipated byU.S. Patent 5268463.

Further, without going into detail here, a well-informed court should hold that claims 1 and 7 are invalid because they fail to meet the requirements of 35 U.S.C. 103, based upon the disclosures of either the Sreekrishna et al. patent (Doc4) or the Liijestroem et al. European patent (Doc5) in combination with the von Schaewen et al. article (Doc6).

G.  Detailed patent information

Patent/application number	Title, Independent Claims and Summary of Claims	Assignee
US 5994629 Earliest priority –  27 Aug 1992 (see terminal disclaimer) Filed – 13 Sep 1995 Granted – 30 Nov 1999 Expected expiry – 27 Aug 2012	Title – Positive selection Claim 1 Genetically transformed plant cells comprisinga desired nucleotide sequence anda co-introduced nucleotide sequencewherein expression or transcription of the co-introduced nucleotide sequence in the transformed cells gives said transformed cells a competitive advantage when a population of cells including the transformed and the non-transformed cells is supplied with a compound that only the transformed cells are able to utilize, and the desired nucleotide sequence codes for a gene other than a toxin, antibiotic or herbicide resistance gene. Claim 4 Genetically transformed maize cells comprisinga desired nucleotide sequence anda co-introduced nucleotide sequencewherein the co-introduced nucleotide sequence gives the transformed cells a competitive advantage when a population of cells including the transformed cells and nontransformed cells is supplied with a compound,wherein the co-introduced nucleotide sequence codes for a phosphomanno-isomerase or a mannophosphatase and the compound is mannose, a mannose derivative or a mannose precursor. Claim 7 A method of selecting genetically transformed cells from a population of cells comprising the steps of:a) introducing into the genome of a plant cell a desired nucleotide sequence and a co-introduced nucleotide sequence wherein said desired nucleotide sequence or co-introduced nucleotide sequence codes for a sequence other than a toxin, antibiotic or herbicide resistance gene;b) obtaining transformed cells;c) supplying to the population of cells a compound that only transformed cells are able to utilize wherein said transformed cells have a competitive advantage over non-transformed cells due to the expression or transcription of the desired nucleotide sequence or co-introduced nucleotide sequence in the presence of the compound; andd) selecting said transformed cells from the population of cells. Claim 27 A method of selecting genetically transformed maize cells from a population of cells comprising the steps of:a) introducing into the genome of a maize cell a desired nucleotide sequence and a co-introduced nucleotide;b) obtaining transformed cells;c) supplying to the population of cells a compound wherein said transformed cells have a competitive advantage over non-transformed cells due to the expression or transcription of the desired nucleotide sequence or co-introduced nucleotide sequence in the presence of the compound; andd) selecting said transformed cells from the population of cells wherein said co-introduced nucleotide sequence comprises a sequence encoding a phosphomanno-isomerase or a manno-phosphatase and the compound is mannose, a mannose derivative or a mannose precursor. This patent is a Continuation in part of US 5767378.	Originally assigned to Novartis AG, and then reassigned to Syngenta Participations AG
EP 530129 A1 Earliest priority –  28 Aug 1991 Filed – 27 Aug 1992 Publication: 3 Mar 1993 Granted – Pending Expected expiry – N/A	Title – Method for the selection of genetically transformed cells and compounds for use in the method Claim 1 A method for selecting from a population of cells genetically transformed cells into which a desired nucleotide sequence has been introduced, wherein in the transformed cells the desired nucleotide sequence or a co-introduced nucleotide sequence induces or increases a positive effect of a compound or nutrient supplied to the population of cells, thereby allowing the transformed cells to be identified or selected from non-transformed cells. Claim 33 Genetically transformed cells whose genome does not contain as a selection marker a non-native nucleotide sequence coding for toxin, antibiotic or herbicide resistance. Claim 35 A compound of the general formula IwhereinR2 is H, CH3, S-CH3, SO2-CH3, SCH2-phenyl, SH, OH, Cl or a group -S-R10, -NH-R10 or -O-R10, where R10 is a β-D-glucopyranuronosyl group or a salt thereof or an ester or amide derivative thereof at the carboxylic acid function,R6 is benzyl which may be substituted on the phenyl ring with OH, C1-6-alkoxy, halogen, C1-4-alkyl, NH2 or CF3, or with -O-R10, -S-R10 or -NH-R10, where R10 is as defined above; C1-8-alkyl or C2-8-alkenyl which may be substituted with from 1 to 3 hydroxy, glucosyloxy or C1-6-alkoxy groups, with phenyl, and/or with -O-R10, -S-R10 or -NH-R10, where R10 is as defined above; esterified C1-6-alkyl or C2-6-alkenyl; furfuryl; or cyclohexylureido, phenylureido or tolylureido;either i) R7 and Y are half-bonds which together form a bond, ii) one of R3 and R9 is H or a group R10 as defined above and the other is a half-bond which together with a half-bond X forms a bond, or R9 is ribosyl, 5 min -phosphoribosyl, glucosyl or -CH2CH(NH2)COOH and R3 is a half-bond which together with the half-bond X forms a bond, and iii) R8 is H, CH3, S-CH3, SO2-CH3, SCH2-phenyl, SH, OH, Cl or a group -S-R10, -NH-R10 or -O-R10, where R10 is as defined above, or iv) R7 is ribosyl, 5 min -phosphoribosyl or glucosyl, R8 is H, R9 and Y are half-bonds which together form a bond, and R3 is a half-bond which together with the half-bond X forms a bond; with the proviso that one of R2, R3, R6, R8 and R9 is or comprises a β-D-glucopyranuronosyl group or a salt thereof or an ester or amide derivative thereof at the carboxylic acid function.	Applicant was Danisco A/S and then reassigned to Sandoz Ltd. and then to Novartis AG
EP 601092 B1 Earliest priority –  27 Aug 1992 Filed – 27 Aug 1992 Granted – 7 Jul 1999 Expected expiry – 27 Aug 2012	Title – Method for the selection of genetically transformed cells and compounds for use in the method Claim 1 A method of selecting genetically transformed plant cells from a population of cells which comprisessupplying the said population with a compound which can be metabolized by the expression product of a nucleotide sequence which has been introduced into the said transformed cells, so as to provide the transformed cells with a physiological advantage when compared to the non-transformed cells, wherein the compound is not an antibiotic or herbicide and has no direct adverse effect on the non-transformed cells. The granted European patent (EP 601092 B1) has significantly reduced number of claims after examination as compared to the original application (EP 601092 A1, which was also published as EP 530129 A1, see above). Opposition from CAMBIA, BASF AG and Unilever PLC was then lodged that led to the further amendment of the claims but the new specification is not available yet. The dates relevant to the opposition are as follows: 31 May 2000 Opposition file by CAMBIA, BASF AG and Unilever PLC 28 Aug 2002 Decision under appeal: Interloculory decision of the Opposition Division of the EPO posted concerning     maintenance of EP 601092 in amended form 21 Jan 2006 Legal effect of interlocutory decision A.106(3) 16 Sep 2006 Fee for printing new specification R.58(5) paid	Applicant was Novartis AG and then reassigned to Syngenta Participations AG
EP 896063 A2 Earliest priority –  28 Aug 1991 Filed – 27 Aug 1992 Publication: 10 Feb 1999 Granted – not as yet Expected expiry – N/A	Title – Method for the selection of genetically transformed cells and compounds for use in the method Claim 1 A plant cell whose genome does not contain as a selection marker an introduced, non-native nucleotide sequence coding for a resistance to a compound having a direct adverse effect on the nontransformed cells such as an antibiotic or herbicide and also not a nucleotide sequence coding for a β-glucuronidase but comprises at least an introduced, non-native nucleotide sequence the expression product of which is capable of providing the transformed plant cell with a physiological advantage when compared to the non-transformed plant cells such that upon supplying a population of plant cells comprising transformed and non-transformed cells with a compound which can be metabolized by the expression product of said introduced, non-native nucleotide sequence, genetically transformed cells can be selected from said population of plant cells. Claim 12 A plant whose genome does not contain as a selection marker an introduced, non-native nucleotide sequence coding for a resistance to a compound having a direct adverse effect on the non-transformed cells such as an antibiotic or herbicide and also not a nucleotide sequence coding for a β-glucuronidase but comprises at least an introduced, non-native nucleotide sequence the expression product of which is capable of providing transformed plant cells derived therefrom with a physiological advantage when compared to the non-transformed plant cells such that upon supplying a population of plant cells comprising transformed and non-transformed cells with a compound which can be metabolized by the expression product of said introduced, non-native nucleotide sequence, genetically transformed cells can be selected from said population of plant cells. This is a divisional application of EP 601092 B1.	Syngenta Participations AG
AU 664200 B2 Earliest priority –  27 Aug 1992 Filed – 27 Aug 1992 Granted – 7 Nov 1995 Expected expiry – 27 Aug 2012	Title – Method for the selection of genetically transformed cells and compounds for use in the method There is no specification available online for this patent.  Claim information will be supplied when possible.	Originally assigned to Sandoz Ltd, and then reassigned to Syngenta Participations AG
Remarks	A PCT application (WO 9305163) was also filed.  Related patents were granted in Canada (CA 2110401), New Zealand (NZ 244135) and Russia (RU 2126834). Application was also filed in Japan (JP 6511146 T2).