DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 1/29/2026 has been entered.
Claim Status
Claims 1-2, 7, 11, 15, 22-23, 25, 29, 32-36, 40, 51-53 and 60 remain pending.
Claims 1, 22-23, 25, 29 and 35 are newly amended.
Claims 1-2, 7, 11, 15, 22-23, 25, 29, 32-36, 40, 51-53 and 60 are examined on the merits.
Response to Applicant Arguments - 35 USC § 103
In response to applicant’s arguments and amendments to the claims dated 01/29/2026 the obviousness rejections of record are withdrawn. However, these amendments necessitate new obviousness rejections which are presented below.
Applicant’s arguments which remain germane to the new grounds of rejection are summarized and addressed below.
These arguments are drawn to the following general concept and specific supporting arguments:
All of the elements of the claims have not been taught or suggested by the prior art and the skilled artisan would not have had a reasonable expectation of success at arriving at the claimed invention upon review of Manela.
Manela is silent on the presence of a second transgene in the transgenic Vitis vinifera cells and given that applicant’s results demonstrate that resveratrol levels did not increase significantly due to overexpression of STS in addition to AroG* while epsilon-viniferin increased the ordinary artisan would not have found it obvious to produce this double mutant.
Applicant urges that given that Manela is silent on epsilon-viniferin concentrations the high concentration of this compound in these cells would not have been predicted from the teachings of Manela.
Manela is silent on the newly added limitations of contact with a phenylalanine solution and a cellular concentration of at least 0.6 mg/g dry weight of epsilon-viniferin.
The office’s argument that arriving at treatment with a range of phenylalanine concentration between about 2mM and 5mM is not persuasive because at least claim 25 is drawn to a specific concentration rather than choosing a concentration within a broad range.
Applicant found unexpected results when producing double transformed cell lines.
Applicant urges that Stilbene levels increased in the double transformed lines compared to the single mutant line. Applicant concludes this argument stating that applicants surprisingly discovered that increasing Phe availability with STS overexpression or FLS overexpression, diverts plant Phe metabolism into the stilbene pathway and/or the flavonoid pathway.
These arguments are not found to be persuasive. First, with respect to a. above, applicant’s argument is not found to be persuasive because while Manela is silent on the presence of a second transgene in the transgenic Vitis vinifera cells, Manela does clearly contemplate modifying transgenic Vitis vinifera cells comprising an AroG* transgene in order to overexpress specific genes directly producing desirable metabolites, explicitly naming the STS and FLS genes. Therefore, while Manela does not state transform the transgenic cells we have created with a STS transgene, given that Manela does teach overexpressing the STS gene in AroG* mutant grape cells it would have been obvious to transform the plants of Manela with a STS transgene in order to overexpress the STS genes in the AroG* background as taught by Manela.
Further, applicants claim a double-transgenic Vitis vinifera cell comprising two elements, first a copy of an AroG* transgene and secondly a STS transgene. Applicant’s urge that the results provided in applicant’s specification teaches away from combining the teachings of Manela to modify FLS and STS in the AroG* background. This is not the case, rather than rely on applicant’s data provided in the specification the obviousness rationale relies on the analysis of whether or not at the time of filing a person having ordinary skill in the art would have found it obvious to combine the teachings to arrive at the claimed invention.
In the instant case, regardless of applicant’s teachings, the rejection of record relies on the teachings of Manela which direct the ordinary artisan to modify the transgenic Vitis vinifera cells comprising the AroG* transgene by further increasing the expression of two specific genes; the STS gene and the FLS gene. Manela even provides motivation to modify the expression of this second gene, noting that grape cells comprising the AroG* gene had higher levels of Resveratrol and a precursor of quercetin, health promoting metabolites and then stating “what will be the effect of overexpressing specific genes directly producing these metabolites, such as STS and FLS…? Will induction of STS further increase resveratrol accumulation in cells in which substrate availability is not limiting” (Manela, Page 22, Last two Paragraphs).
Therefore, because Manela teaches grape cells which differ from the claimed inventio by only a single element and that Manela teaches motivation to introduce that element into the cells rather than picking and choosing elements the rejection follows the teachings of Manela to arrive at the claimed invention.
With respect to the argument described in a. i. while Manela is silent on the concentration of epsilon-viniferin, Tříska makes clear that epsilon-viniferin concentrations greater than that required by the instant invention are intrinsic characteristics of cells from the stems of grape plants and therefore, applicant’s arguments that because Manela is silent on this specific compound the ordinary artisan would not have been motivated to combine the teachings of Manela to arrive at cells having increased concentrations of desirable metabolites is not persuasive. The ordinary artisan would still have been motivated to combine these teachings to produce increased concentrations of other important metabolites. It is important to note that epsilon-viniferin is a member of a class of desirable metabolites produced from resveratrol and Manela does teach elevated levels of resveratrol and therefore this motivation would not exclude elevated levels of viniferin even though Manela does not explicitly teach elevated epsilon-viniferin levels.
With respect to applicant’s arguments summarized in b. above, applicant’s arguments are not found to be persuasive because Li and Aghdan make clear that feeding 5.0 mM phenylalanine to a variety of different systems including yeast cells and tomato cells produces high levels of desirable metabolites, which are higher than those produced when cells were fed with lower concentrations of phenylalanine. Therefore it would have been obvious to use this specific concentration of phenylalanine and applicant’s arguments are not found to be persuasive.
Applicant’s arguments summarized in c. above are not found to be persuasive because while applicant urges that there were unexpected results, these results appear to be expected given the teachings of Manela.
Specifically, the ordinary artisan would expect that increasing the expression of an enzyme which uses Phe and Tyr to produce Resveratrol (a stilbene) would result in the production of more stilbenes.
When determining if an invention provides an unexpected result the evidence of unexpected and expected properties must be weight. The MPEP in 716.02 (C) I, discloses that “Evidence of unexpected results must be weighed against evidence supporting prima facie obviousness in making a final determination of the obviousness of the claimed invention”. This section continues stating “Where the unexpected properties of a claimed invention are not shown to have a significance equal to or greater than the expected properties, the evidence of unexpected properties may not be sufficient to rebut the evidence of obviousness”. Finally in 716.02 (c) II, the MPEP states that “Expected beneficial results are evidence of obviousness of a claimed invention, just as unexpected results are evidence of unobviousness thereof”.
With respect to the instant case, given the teachings of Manela it would have been obvious to increase the expression of the STS gene in the AroG* mutant grape lines given that Manela states this explicitly. This weighs heavily in the case of obviousness. Further, applicant’s evidence of an unexpected result appears to be that the levels of stilbenes were higher in cells comprising an additional STS gene. Rather than being unexpected, given the teachings of Manela, this appears to be the expected result. For example, the evidence in Manela teaches that the rate limiting step in Stilbene synthesis is the production of PHE and TYR, that AroG* mutant grape cells produce higher levels of these metabolites than wild-type cells and that STS converts these precursors into Stilbenes and that Manela then suggests overexpressing STS in AroG* transgenic grape cells. Given this evidence it appears that transforming the AroG* mutant cells with STS would have the obvious result of increasing stilbenes.
Further, applicant urges that Phenylalanine feeding of these cells produced higher levels of stilbenes and flavonoids as compared to feeding of the AroG* line alone and that this is unexpected. This is not unexpected given that Manela states explicitly that “concentrations of AAAs, and of phenylalanine in particular, are rate-limiting in production of these metabolites”, which makes clear that increasing the concentration of phenylalanine would increase the amount of these metabolites produced (Manela, Page 1, Abstract).
Thus, there appears to be more evidence of obviousness than unexpected results, the unexpected properties are not shown to have greater significance than expected properties, and finally that expected beneficial results are evidence of obviousness. Given this, applicant’s limited evidence of unexpected results is insufficient to overcome the obviousness of the claimed invention given the teachings of Manela and this argument is not found to be persuasive.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Applicant’s amendment to the claims filed on 1/29/2026 has necessitated this new ground of rejection. Specifically, the amendment of claim 1 to include limitations drawn to contacting the cell with phenylalanine and wherein the cell comprises a concentration of at least 0.6 mg/g dray weight epsilon-viniferin. This amendment requires a new analysis to appropriately address these new limitations. Accordingly, this new ground of rejection is being presented below.
Claims 1-2, 7, 11, 15, 22-23, 25, 29, 32-36, 40 and 60 are newly rejected under 35 U.S.C. 103 as being obvious under by Manela as evidenced by Tříska, Molecules 22.6 (2017): 928 and in view of Li, Scientific reports 6.1 (2016): 36827 and Aghdam, Scientia Horticulturae 246 (2019): 818-825.
With respect to claim 1, Manela teaches a transgenic Vitis vinifera cell comprising an Agrobacterium tumafaciens introduced copy of an AroG* gene (Manela, Page 1, Lines 5-10).
Further, Manela teaches that stilbene synthase (STS) is a key enzyme in the production of stilbenoids and provides a summary of some of the metabolic pathways in grape cells which include native STS and flavanol hydroxylase genes (Manela, Page 11, Figure 9; Manela, Page 7, Column 1, Only Paragraph). Manela also notes that resveratrol and quercetin are both metabolite that were increased in AroG* lines and that these compounds are considered health promoting metabolites due to their anti-oxidative characteristics and ability induce so-called longevity genes which can protect humans from age-related diseases.
While Manela notes that in their experiment the results suggest that the availability of Phe and Tyr is a rate limiting factor in the production of those metabolites in Gamay cell culture, an interesting question for further understanding how the production of these metabolites is controlled in grapes is what is the effect of overexpressing the genes directly producing these metabolites, such as STS and FLS which produce resveratrol and quercetin respectively in AroG* cells? Manela further wonders, “will induction of STS further increase resveratrol accumulation in cells in which substrate availability is not limiting” (Manela, Page 11, Columns 1-2; See also Manela, Page 11, Figure 9).
To clarify, Manela teaches four Vitis vinifera cell lines which comprise independent insertions with a feedback-insensitive bacterial DAHPS (AroG*) gene in suspension culture. Manela examined metabolite and gene expression profiles for these cells and noticed that they had elevated levels of several important secondary metabolites including resveratrol and quercetin which have desirable health promoting properties. Manela further noted that given their work it appeared that the rate limiting step in producing these compounds in their cell culture was the presence of the precursor molecules but that the STS and FLS enzymes are responsible for directly producing those metabolites. Manela then teaches motivation for introducing a second transgene into these cells that encodes for a STS or FLS enzyme stating “What will be the effect of overexpressing specific genes directly producing these metabolites, such as STS and FLS for resveratrol land quercetin respectively, in AroG* cells? Will induction of STS further increase resveratrol accumulation in cells in which substrate availability is not limiting?”
Finally, Manela teaches motivation to contact these cells with exogenous phenylalanine stating that the concentration of aromatic amino acids and in particular phenylalanine are rate-limiting in the production of specialized metabolites downstream of the shikimate and aromatic amino acid pathways which include the stilbenes like the viniferins (Manela, Page 1, Abstract; Manela, Page 11, Figure 9).
While, Manela does not explicitly state the epsilon-viniferin concentration in the transgenic plants comprising the AroG* gene and does not provide a level for the contemplated double mutant having increased FLS or STS in addition to the AroG* gene, epsilon-viniferin concentrations of grape cane cells are known in the art. Specifically, Tříska provides evidence of an intrinsic characteristic of grape plants; the concentration of specific metabolites in these cells. Of particular relevance, Tříska, in Figure 2, provides stem trans-epsilon-viniferin concentrations for seven different grape varieties, each grown in three different locations with measurements taken for three consecutive years (Tříska, Page 4, Figure 2). The lowest of all of these measurements is between 500 and 1000 mg/kg and the material and methods section on pages 8-9 makes clear that these measurements were recorded in dry weight (Tříska, Section 4.3, Pages 8-9). Given that 500 mg/kg is 0.5 mg/g and that the lowest measurement provided in the extensive sampling of Tříska is well above this mark with many other samples having concentrations above 3mg/g it appears that grape cells intrinsically have a epsilon-viniferin concentration of greater than about 0.6 mg/g.
With respect to claim 1, Manela does not explicitly teach a Vitis vinifera cell comprising a second transgene comprising a stilbene synthase gene or a flavanol synthase gene nor does Manela explicitly teach contacting the cell with phenylalanine in a concentration of about 2mM to about 5mM where the cell comprises a concentration of at least 0.6 mg/g dry weight epsilon-viniferin.
With respect to claim 1, Li and Aghdam teach the benefits of 5mM phenylalanine feeding in the production of phenols, flavonoids and anthocyanins in engineered yeast and in tomato fruits (Li, Page 1, Abstract; Li, Page 2, First and Third Paragraphs of Results; Aghdam, 818 Abstract). Specifically, these two references teach that this higher concentration of phenylalanine lead to higher levels of desirable secondary metabolites including resveratrol the precursor to other stilbenes including the viniferins (Li, Page 2, Last Sentence of third paragraph of results; Aghdam, Page 818, Abstract; Manela, Page 11, Figure 9).
At the time of filing it would have been obvious to the ordinary artisan given the teachings of Manela to modify the Vitis vinifera cells of Manela by introducing a second transgene encoding a STS or FLS enzyme in order to overexpress those specific genes in an environment where substrate availability was not rate limiting in order to produce higher levels of resveratrol or quercetin in Vitis vinifera cells. As such it would have been obvious to generate these cells and then to treat them with exogenous phenylalanine in order to overcome the rate limiting factor limiting the production of specialized metabolites in grape cells through the shikimate and aromatic amino acid biosynthetic pathways.
This would be obvious because Manela suggests overexpressing STS and/or FLS in cells where a transgene encoding an AroG* gene was already introduced and which enhanced the production of desirable metabolites. Further, Manela makes clear in the abstract that phenylalanine is the rate limiting factor in the production of these desirable metabolites. Further, using a concentration of 5 millimolar phenylalanine would have been obvious given the teachings of Li and Aghdam which demonstrate that feeding with phenylalanine at a concentration of 5 mM produced higher levels of desirable secondary metabolites compared to lower concentrations. Finally, Tříska demonstrates that epsilon-viniferin concentrations greater than 0.6 mg/g dry weight are an intrinsic characteristic of grape cells and given the modifications to these grape cells the epsilon-viniferin content would be even greater.
The ordinary artisan would have been motivated to generate these Vitis vinifera cells because while the AroG* cells produce elevated levels of heath promoting metabolites it appears that the rate limiting step for producing these metabolites is the amount of substrate.
Given this information the ordinary artisan would have been motivated to introduce the STS and FLS enzymes that are directly responsible for this metabolite production into AroG* cells to determine if this was capable of producing more of the metabolites alone and further the ordinary artisan would have been motivated to supplement these cells with the Phe and Tyr substrates. While performing this supplementation the ordinary artisan would have been motivated to optimize experimental conditions to lead to the highest level of secondary metabolite production and therefore would have supplemented the cells with phenylalanine in a concentration of 5mM given the results of Li and Aghdam that demonstrate that in two different systems this concentration produced higher levels of secondary metabolites than feeding with other, lower concentrations of phenylalanine.
This would have been motivating because it would allow for the production of grape cells having the highest concentration of resveratrol, quercetin, epsilon-viniferin and other metabolites which have desirable anti-oxidative, health promoting characteristics. Grape cells containing the highest level of these metabolites would be valuable in producing these metabolites but also for imparting health promoting properties in products produced from grapes.
Therefore, it would have been obvious to the ordinary artisan at the time of filing to modify the plants of Manela as suggested by Manela to produce grape cells comprising a second transgene encoding a STS or FLS enzyme and to treat these cells with exogenous phenylalanine at a concentration of 5mM as taught in Li or Aghdam, given the motivation provided in Manela which would have produced cells having the claimed epsilon-viniferin concentrations given the evidence of Tříska and claim 1 is rejected as obvious.
With respect to claim 2, Manela as evidenced by Tříska in view of Li and Aghdam teaches all of the limitations of claim 1, see above. Further, Manela teaches where the cells are Vitis vinifera cv. Gamay Red cells (Manela, Page 1, Line 6).
With respect to claim 7, Manela as evidenced by Tříska in view of Li and Aghdam teaches all of the limitations of claim 1, see above. Further, Manela teaches the STS gene which would encode the STS enzyme which is the key enzyme in production of stilbenoids (Manela, Page 7, Column 1, Lines 6-8; Manela, Page 11, Figure 9).
With respect to claim 11, Manela as evidenced by Tříska in view of Li and Aghdam teaches all of the limitations of claim 1, see above. Further, Manela teaches that quercetin is a flavanol produced by the flavonoid pathway by the enzyme Flavonoid 3’ hydroxylase (F3’h), which Manela refers to as FLS (Manela, Page 2, Column 1, Last Three lines; Manela, Page 11, Figure 9; Manela, Page 11, Column 2, Lines 1-3).
With respect to claim 15, Manela as evidenced by Tříska in view of Li and Aghdam teaches all of the limitations of claim 1, see above. Further, Manela teaches that the AroG* gene is found in a construct comprising a 35s CaMV constitutive promoter and therefore the AroG* gene is functionally-linked to a constitutive promoter (Manela, Page 3, Column 2, First Full Paragraph).
With respect to claim 22, Manela as evidenced by Tříska in view of Li and Aghdam teaches all of the limitations of claim 1, see above. Further, Manela teaches that AroG* transgenic cells comprise a higher level of aromatic amino acids (AAA) including phenylalanine and tyrosine (Manela, Page 1, Lines 10-12).
With respect to claim 23, Manela as evidenced by Tříska in view of Li and Aghdam teaches all of the limitations of claim 1, see above. Further, Manela teaches transgenic cells comprising a more than 20 fold increase in resveratrol compared to control cells (Manela, Page 1; Manela, Page 8, Figure 6, Panel E). While Manela is silent on the dry weight of resveratrol and its concentration in mg/g given this increase in resveratrol concentration it is clear that this is an inherent property of the transgenic cells of Manela. This is supported by applicants own data in figure 3 which demonstrates that the highest fold change in resveratrol occurs in AroG* transformed lines like the cells of Manela and in figure 7A which shows that the base level of resveratrol in Vitis vinifera cv. Gamay Red cells is around 0.25 mg/g dry weight. Given that the cells of Manela are also Vitis vinifera cv. Gamay Red cells the 0.25 mg/g dry weight of resveratrol in these cells is an inherent property and as noted above, given that the transformed cells of Manela demonstrated a more than 20 fold increase in resveratrol these cells would comprise a dry weight of resveratrol of around 5mg/g resveratrol.
Therefore, as outlined above a dry weight of at least 0.8 mg/g resveratrol is an inherent property of the transgenic Vitis vinifera cells of Manela.
With respect to claim 25, Manela as evidenced by Tříska in view of Li and Aghdam teaches a transgenic Vitis vinifera cell comprising an Agrobacterium tumafaciens introduced copy of an AroG* gene (Manela, Page 1, Lines 5-10).
Further, Manela teaches that stilbene synthase (STS) is a key enzyme in the production of stilbenoids and provides a summary of some of the metabolic pathways in grape cells which include native STS and flavanol hydroxylase genes (Manela, Page 11, Figure 9; Manela, Page 7, Column 1, Only Paragraph). Manela also notes that resveratrol and quercetin are both metabolite that were increased in AroG* lines and that these compounds are considered health promoting metabolites due to their anti-oxidative characteristics and ability induce so-called longevity genes which can protect humans from age-related diseases.
While Manela notes that in their experiment the results suggest that the availability of Phe and Tyr is a rate limiting factor in the production of those metabolites in Gamay cell culture, an interesting question for further understanding how the production of these metabolites is controlled in grapes is what is the effect of overexpressing the genes directly producing these metabolites, such as STS and FLS which produce resveratrol and quercetin respectively in AroG* cells? Manela further wonders, “will induction of STS further increase resveratrol accumulation in cells in which substrate availability is not limiting” (Manela, Page 11, Columns 1-2; See also Manela, Page 11, Figure 9). This final sentence indicates that not only does Manela provide motivation to generate AroG* grape cells comprising a second transgene encoding a STS or FLS enzyme but they contemplate assaying metabolite profiles under conditions where substrate is not limiting. In order to have conditions where substrate is not limiting the grape cells would have to be treated with phenylalanine and tyrosine which are the substrates Manela teaches can be rate limiting (Manela, Page 11, Column 1).
With respect to claim 29, Manela as evidenced by Tříska in view of Li and Aghdam teaches all of the limitations of claim 25 taught above, see above.
With respect to claim 32, Manela as evidenced by Tříska in view of Li and Aghdam teaches all of the limitations of claim 1, see above. Further, paragraph 00048 on page 8 of the instant specification makes clear that a pharmaceutical composition can comprise a doubly-transgenic Vitis vinifera cell or an extract or fraction thereof and the specification provides no further guidance on what is required for a composition to be a pharmaceutical composition and therefore it is clear that the transgenic cells of Manela are a pharmaceutical composition.
With respect to claim 33, Manela as evidenced by Tříska in view of Li and Aghdam teaches all of the limitations of claim 32 taught above, see above. Further, Manela teaches all of the limitations of claim 25 taught above, see above.
With respect to claim 34, Manela as evidenced by Tříska in view of Li and Aghdam teaches all of the limitations of claim 25 taught above, see above.
With respect to claim 35, Manela as evidenced by Tříska in view of Li and Aghdam teaches all of the limitations of claim 32 taught above, see above. Further, Manela teaches that the transgenic grape cells comprise elevated levels of resveratrol and therefore the composition of claim 32 includes resveratrol (Manela, Page 1; Manela, Page 8, Figure 6, Panel E).
With respect to claim 36, Manela as evidenced by Tříska in view of Li and Aghdam teaches all of the limitations of claim 32 taught above, see above. Further, Manela teaches extracts from liquid cultured cells including transgenic cell lines which were then used for immunoblot analysis (Manela, Page 3, Column 2, Second full paragraph; Manela, Page 5, Figure 2).
With respect to claim 40, Manela as evidenced by Tříska in view of Li and Aghdam teaches all of the limitations of claim 32 taught above, see above. Further, Manela teaches extracts from liquid cultured cells including transgenic cell lines which were then used for immunoblot analysis, these extracts were produced by grinding samples of the cell culture in liquid nitrogen, this solution was then centrifuged to separate soluble protein extracts from cell precipitates, this would produce an extract substantially devoid of intact and ruptured cells (Manela, Page 3, Column 2, Second full paragraph; Manela, Page 5, Figure 2).
With respect to claim 60, Manela as evidenced by Tříska in view of Li and Aghdam teaches all of the limitations of claim 32 taught above, see above. These extracts are grape plant cells and are therefore edible, this is an inherent property of grape plant cells.
With respect to claims 2, 7, 11, 15, 22-23, 25, 29, 32-36, 40 and 60, Manela as evidenced by Tříska does not explicitly teach a Vitis vinifera cell comprising a second transgene comprising a stilbene synthase gene or a flavanol synthase gene nor does Manela explicitly teach contacting the cell with phenylalanine in a concentration of about 2mM to about 5mM where the cell comprises a concentration of at least 0.6 mg/g dry weight epsilon-viniferin.
With respect to claims 2, 7, 11, 15, 22-23, 25, 29, 32-36, 40 and 60, Li and Aghdam teach the benefits of 5mM phenylalanine feeding in the production of phenols, flavonoids and anthocyanins in engineered yeast and in tomato fruits (Li, Page 1, Abstract; Li, Page 2, First and Third Paragraphs of Results; Aghdam, 818 Abstract). Specifically, these two references teach that this higher concentration of phenylalanine lead to higher levels of desirable secondary metabolites including resveratrol the precursor to other stilbenes including the viniferins (Li, Page 2, Last Sentence of third paragraph of results; Aghdam, Page 818, Abstract; Manela, Page 11, Figure 9).
At the time of filing it would have been obvious to the ordinary artisan given the teachings of Manela to modify the Vitis vinifera cells of Manela by introducing a second transgene encoding a STS or FLS enzyme in order to overexpress those specific genes in an environment where substrate availability was not rate limiting in order to produce higher levels of resveratrol or quercetin in Vitis vinifera cells. As such it would have been obvious to generate these cells and then to treat them with exogenous phenylalanine in order to overcome the rate limiting factor limiting the production of specialized metabolites in grape cells through the shikimate and aromatic amino acid biosynthetic pathways.
This would be obvious because Manela suggests overexpressing STS and/or FLS in cells where a transgene encoding an AroG* gene was already introduced and which enhanced the production of desirable metabolites. Further, Manela makes clear in the abstract that phenylalanine is the rate limiting factor in the production of these desirable metabolites. Further, using a concentration of 5 millimolar phenylalanine would have been obvious given the teachings of Li and Aghdam which demonstrate that feeding with phenylalanine at a concentration of 5 mM produced higher levels of desirable secondary metabolites compared to lower concentrations. Finally, Tříska demonstrates that epsilon-viniferin concentrations greater than 0.6 mg/g dry weight are an intrinsic characteristic of grape cells and given the modifications to these grape cells the epsilon-viniferin content would be even greater.
The ordinary artisan would have been motivated to generate these Vitis vinifera cells because while the AroG* cells produce elevated levels of heath promoting metabolites it appears that the rate limiting step for producing these metabolites is the amount of substrate.
Given this information the ordinary artisan would have been motivated to introduce the STS and FLS enzymes that are directly responsible for this metabolite production into AroG* cells to determine if this was capable of producing more of the metabolites alone and further the ordinary artisan would have been motivated to supplement these cells with the Phe and Tyr substrates. While performing this supplementation the ordinary artisan would have been motivated to optimize experimental conditions to lead to the highest level of secondary metabolite production and therefore would have supplemented the cells with phenylalanine in a concentration of 5mM given the results of Li and Aghdam that demonstrate that in two different systems this concentration produced higher levels of secondary metabolites than feeding with other, lower concentrations of phenylalanine.
This would have been motivating because it would allow for the production of grape cells having the highest concentration of resveratrol, quercetin, epsilon-viniferin and other metabolites which have desirable anti-oxidative, health promoting characteristics. Grape cells containing the highest level of these metabolites would be valuable in producing these metabolites but also for imparting health promoting properties in products produced from grapes.
Therefore, it would have been obvious to the ordinary artisan at the time of filing to modify the plants and methods of Manela as suggested by Manela to produce grape cells comprising a second transgene encoding a STS or FLS enzyme and to treat these cells with exogenous phenylalanine at a concentration of 5mM as taught in Li or Aghdam, given the motivation provided in Manela which would have produced cells having the claimed epsilon-viniferin concentrations given the evidence of Tříska and claims 2, 7, 11, 15, 22-23, 25, 29, 32-36, 40 and 60 are rejected as obvious.
Claims 51-53 are newly rejected under 35 U.S.C. 103 as being unpatentable over Manela as evidenced by Tříska in view of Li, Aghdam and Rieder.
With respect to claim 51, Manela as evidenced by Tříska in view of Li and Aghdam teaches all of the limitations of claim 32, see above.
With respect to claim 51, Manela as evidenced by Tříska in view of Li and Aghdam does not teach a method of treating a Cytokine Storm in a patient in need, comprising administering to the patient a therapeutically-effective amount of the composition of claim 32.
With respect to claim 51, Rieder teaches that resveratrol treatment attenuated the cytokine storm in mice (Rieder, Page 1244, Third Paragraph). Further, Rieder contemplates administering this to human patients, noting that this treatment may constitute a novel therapeutic modality to prevent and treat lung inflammation by acting through several pathways to reduce pulmonary infection (Rieder, Page 1244, Fourth Paragraph).
At the time of filing it would have been obvious to use the composition of claim 32 to treat patients having an incidence of a Cytokine Storm by administering to the patient the composition of claim 32. This would have been obvious because the composition of claim 32 is composed of grape cells having elevated resveratrol levels and the treatment method of Rieder comprises administering resveratrol to patients. Therefore administering the grape cells directly to patients is an efficient method of treating patients with resveratrol and would be motivating to the ordinary artisan as it does not require extensive isolation steps and allows for the delivery a much higher dose of resveratrol than would be possible using the same amount of wild type grape cells. Therefore it would have been obvious to combine the teachings of Manela as evidenced by Tříska in view of Li and Aghdam with those of Rieder and claim 51 is rejected as obvious.
With respect to claims 52 and 53, Manela as evidenced by Tříska in view of Li, Aghdam and Rieder collectively teach all of the limitations of claim 51, see above. Further, the combined method of these references is effective in treating cytokine storm and therefore would have been effective in treating the cytokine storm associated with Coronavirus infection including Severe acute respiratory syndrome coronavirus 2 infection. Therefore, claims 52-53 are rejected as obvious under Manela as evidenced by Tříska in view of Li, Aghdam and Rieder.
Conclusion
All claims are rejected.
Contact Information
Any inquiry concerning this communication or earlier communications from the examiner should be directed to BRIAN JAMES SULLIVAN whose telephone number is (571)272-0561. The examiner can normally be reached 7:30 to 5:00.
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/BRIAN JAMES SULLIVAN/ Examiner, Art Unit 1663
/Amjad Abraham/SPE, Art Unit 1663