DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application is being examined under the pre-AIA first to invent provisions.
Response to Amendment
Applicant's request for reconsideration of the Non-Final of the rejection of the last Office action is persuasive and, therefore, the Non-finality of that action is withdrawn.
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 pre-AIA 35 U.S.C. 103(a) which forms the basis for all obviousness rejections set forth in this Office action:
(a) A patent may not be obtained though the invention is not identically disclosed or described as set forth in section 102, if the differences between the subject matter sought to be patented and the prior art are such that the subject matter as a whole would have been obvious at the time the invention was made to a person having ordinary skill in the art to which said subject matter pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under pre-AIA 35 U.S.C. 103(a) are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims under pre-AIA 35 U.S.C. 103(a), the examiner presumes that the subject matter of the various claims was commonly owned at the time any inventions covered therein were made absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and invention dates of each claim that was not commonly owned at the time a later invention was made in order for the examiner to consider the applicability of pre-AIA 35 U.S.C. 103(c) and potential pre-AIA 35 U.S.C. 102(e), (f) or (g) prior art under pre-AIA 35 U.S.C. 103(a).
Claims 27-39 are rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Koprowski et al. (US 2010/0184130 A1).
Koprowski teaches genetically modified plants having green biomass (defined as leaves and stems) with an increased amount of oil in green biomass relative to the non-genetically modified counterpart (see, e.g., ¶¶[0002], [0007], [0027]–[0034]). Koprowski discloses that such modification can increase oil accumulation in tobacco green biomass (tobacco leaves) to 5–8% of dry weight (¶[0131]), which falls within the claimed range of 5–25% (w/w dry weight) non-polar lipid. Koprowski further teaches that the genetically modified plant is engineered to have increased expression of (i) a first gene encoding an enzyme involved in oil biosynthesis such as an acyl transferase, expressly including acyl CoA:diacylglycerol acyltransferase (DGAT) (¶¶[0014], [0040]), and (ii) a second gene encoding a transcription factor that regulates seed development, wherein the transcription factor is selected from LEC1, LEC2, FUS3 and WR1 (¶¶[0015], [0045]). Koprowski further discloses that the acyl transferase enzyme and transcription factor are co-expressed in the same cells of the green biomass (¶¶[0016]–[0018], [0046]–[0049], [0051]–[0058]).
Thus, Koprowski teaches transgenic vegetative plant parts (green biomass such as leaves) which comprise exogenous polynucleotides encoding DGAT and a transcription factor selected from the group consisting of LEC1, LEC2, FUS3 and WR1 (i.e., WRI1) and which have an oil (triacylglycerol / total fatty acid) content of about 5–8% of dry weight, within the claimed 5–25% range.
Koprowski further teaches producing biodiesel oil by extracting oil from such plants transformed according to this invention and converting the oils to biofuels (¶¶[0002], [0060]–[0063]). Koprowski describes both physical (mechanical) extraction methods (expeller, screw press, ram press) and solvent extraction from dried/ground green biomass with organic solvents such as hexane, followed by separation and recovery of the oil from the solvent by distillation or similar means (¶¶[0060]–[0061]). Accordingly, Koprowski teaches (i) a collection of transgenic vegetative plant parts (green biomass, e.g., tobacco leaves) comprising exogenous polynucleotides encoding DGAT and a transcription factor including WR1 (WRI1); (ii) such biomass having 5–8% oil by dry weight, within the claimed 5–25% range; (iii) extracting lipid from that biomass by mechanical and/or solvent extraction; and (iv) recovering the extracted lipid. To the extent claim 27 is argued to require the specific combination of WRI1 + DGAT rather than any transcription factor from the enumerated set, the use of WRI1 (WR1) is expressly taught as one of the transcription factors to be employed together with DGAT in green biomass (¶¶[0015], [0045], [0046]; see also the generic description that the first and second genes are co-expressed in green tissue). Selecting WRI1 from the explicitly disclosed list of alternative seed-development transcription factors (LEC1, LEC2, FUS3, WR1) in Koprowski would have been no more than the predictable substitution of one known, functionally equivalent transcription factor for another in a system expressly designed to increase oil accumulation in green biomass, with a reasonable expectation of success in obtaining high-oil vegetative tissues. See KSR Int’l Co. v. Teleflex Inc., 550 U.S. 398 (2007).
It would have been obvious to one of ordinary skill in the art at the time of the invention to employ WR1 (WRI1) instead of LEC2 in Koprowski’s DGAT + TF system to generate transgenic green biomass having elevated oil content in the 5–25% range, and to extract and recover lipid from such biomass using the extraction methods already disclosed by Koprowski.
Regarding Claim 28
Koprowski explicitly teaches physical extraction methods, including mechanical extraction by expeller, screw press, and ram press (¶[0060]), which are forms of pressing/crushing and necessarily involve mechanical comminution of the biomass. Koprowski also teaches solvent extraction by mixing an organic solvent (e.g., hexane) with dried and ground green biomass, followed by separation of the oil/solvent phase from the biomass (¶[0061]). Thus, all of the options recited in claim 28 are explicitly taught by Koprowski.
Regarding Claim 29
Koprowski expressly teaches hexane as a typical organic solvent for extraction of oil from the dried and ground green biomass (¶[0061]) and also references “modified hexane extraction or classic chloroform-methanol isolation” of fatty acids in Example 5 (¶[0125]). These match the claimed hexane and chloroform/methanol solvents. The use of other conventional organic solvents for lipid extraction such as diethyl ether, petroleum ether, butanol, or benzene would have been an obvious routine choice from among known oil-extraction solvents that are functionally interchangeable, providing no patentable distinction.
Regarding Claim 30
Koprowski describes that oils extracted from genetically modified plants of the invention are used to produce biodiesel fuel and other industrial biofuel oils (¶¶[0002], [0006], [0060]–[0063]). Koprowski describes transesterification of extracted plant oil with methanol in the presence of sodium hydroxide (a chemical catalyst), optionally preceded by alkali refining (¶[0062]). Transesterification is a chemical process often conducted at elevated temperature and is a paradigmatic example of “chemical means” (and often includes heat); enzymatic transesterification is also well known in the art and would be an obvious alternative to chemical catalysis. Thus, converting the extracted lipid to an industrial product (such as biodiesel) via heat and/or chemical or enzymatic treatment is taught or would have been obvious in view of Koprowski. Claim 30 is therefore obvious.
Regarding Claim 31
Koprowski expressly teaches producing biodiesel from extracted plant oils by transesterification, wherein the vegetable oil is reacted with methanol (an alcohol) in the presence of sodium hydroxide to produce methyl esters (biodiesel) and glycerin (¶[0062]). Methyl esters are alkyl esters of fatty acids.
Regarding Claim 32
Koprowski teaches that biodiesel is produced by reacting the extracted plant oil with methanol in the presence of sodium hydroxide, which is a chemical catalyst, as part of the transesterification reaction (¶[0062]). Koprowski further notes that biodiesel produced from vegetable oils can partially substitute petroleum diesel fuel and is used as a substitute for or blend with diesel under ASTM standards (¶[004], [0062]). Thus, It would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to have modified the process of Koproski by using a catalyst in the reaction with an alcohol and blending the resulting alkyl esters with petroleum-based fuel would have been obvious in view of Koprowski.
Regarding Claim 33
Koprowski describes conventional refining of plant oils prior to biodiesel production, referencing “alkali refining” to remove impurities, which includes processes such as degumming and removal of phospholipids, waxes, and colored or odorous components (¶[0062]). Koprowski also quantifies the fatty acid composition of extracted oils by gas chromatography (GC) and LC-MS (¶¶[0125]–[0129]), comparing relative amounts of oleic, linoleic, linolenic and palmitic acids. Such refining steps (degumming, deodorizing, decolorizing, drying, fractionating, wax removal) and analysis of fatty acid composition are standard and routine in the edible oil and biodiesel industries and would have been obvious to apply to oils extracted from the high-oil green biomass of Koprowski.
Regarding Claim 34
Koprowski discloses that in DGAT-overexpressing plants, and in plants further modified with seed-development transcription factors, the fatty acid composition of extracted fatty acid esters is altered. In the transgenic tobacco lines, the proportion of oleate (18:1) is increased from about 1.5% to 20–25% of total extracted fatty acids, while linolenate (18:3) is reduced from ~50–60% to ~30–40%, and significant amounts of palmitate (16:0) and linoleate (18:2) remain present (¶¶[0125]–[0129]; see also FIGS. 5 and 6B). Koprowski also notes that these changes in fatty acid composition make tobacco biomass oil more similar to canola oil and desirable for biodiesel (¶[0130]). Thus, Koprowski teaches or suggests vegetative plant parts whose non-polar lipid has oleic acid at ~20–25%, reduced linolenic acid, and significant fractions of palmitic and linoleic acids. The claimed thresholds of ≥19% oleic and <15% α-linolenic acid, as well as the specified minimums for palmitic and linoleic acids, represent optimization and selection of particular compositional windows within the ranges that would have been obtained or readily achievable by adjusting the expression levels or combinations of DGAT and transcription factors in Koprowski’s system. Selecting specific compositional cut-offs that fall within or are readily attainable based on the trends disclosed in Koprowski would have been obvious to a person of ordinary skill seeking to further optimize oil quality for biodiesel performance.
Regarding Claim 35
Koprowski reports that in DGAT-overexpressing lines, the percentage of oleate increases from ~1.5% in wild-type tobacco to 20–25% in modified plants (¶[0128]), which is well above a 2% increase. Koprowski also shows substantial shifts in the composition of other fatty acids (including palmitate and linoleate) relative to the wild-type control (¶¶[0125]–[0129], FIG. 6B). Accordingly, the recited requirement of at least 2% more oleic acid (and/or at least 2% less palmitic acid) relative to the corresponding wild-type vegetative plant part is fully consistent with, and would have been obvious in light of, the compositional shifts reported by Koprowski.
Regarding Claim 36
Koprowski teaches substantially altering lipid metabolism in green biomass by overexpressing DGAT and seed-development transcription factors, thereby redirecting carbon flow into triacylglycerol and changing the composition of storage and membrane lipids (¶¶[0007], [0035]–[0040], [0125]–[0129]). It is well understood in the art that such major perturbations of lipid biosynthesis and storage can affect not only triacylglycerols and fatty acyl composition but also other lipid classes including sterols, sterol esters, and sterol glycosides. Given the scope of lipid metabolic engineering disclosed by Koprowski, and the explicit goal of increasing oil accumulation at the expense of other components in green biomass, it would have been obvious that levels of sterols and related sterol derivatives in the non-polar lipid fraction would be modified relative to wild-type, and that such changes could be monitored or exploited as desired.
Regarding Claim 37
Koprowski explains that the plant oils of interest are vegetable oils, particularly triacylglycerols (TAG), and that the target is increased oil accumulation in green biomass with TAG as the main storage form (¶¶[0006], [0032], [0034], [0038], [0040]). Example 5 shows that in DGAT-overexpressing tobacco lines, the TAG fraction of the tobacco biomass is increased 3–7-fold, and the TAG fraction is analyzed by LC-MS as the dominant neutral lipid species (¶¶[0125]–[0127], FIG. 4, FIG. 5). It would have been obvious for a skilled artisan, in implementing Koprowski’s teaching to maximize TAG accumulation in green biomass, to select or further engineer lines in which TAG constitutes a very high proportion of the non-polar lipid fraction (e.g., ≥ 90%), as a straightforward optimization goal in high-TAG green biomass lines.
Regarding Claim 38
As discussed above, Koprowski teaches co-expression in green biomass of a first gene encoding an acyl transferase, including DGAT, with a second gene encoding a seed-development transcription factor selected from the group LEC1, LEC2, FUS3, WR1 (WRI1) (¶¶[0014]–[0017], [0040], [0045]–[0048], [0051], [0056]–[0058]). Koprowski further teaches that such combinations substantially increase TAG accumulation and total extractable fatty acids in green biomass (¶¶[0011], [0038], [0125]–[0132]). A person of ordinary skill in the art would have reasonably expected that co-expressing a transcription factor that activates seed-oil biosynthetic programs (e.g., WR1/WRI1) together with DGAT (which catalyzes TAG assembly) would produce at least an additive, and likely more-than-additive, increase in non-polar lipid accumulation relative to either factor expressed alone, since one gene increases substrate supply and the other increases TAG assembly capacity. Selecting WRI1 from Koprowski’s enumerated set, and recognizing that the observed increase in TAG accumulation in such co-expression lines is greater than the simple sum of effects observed for each single gene, would have been an inherent and predictable consequence of implementing Koprowski’s co-expression strategy. As such, the “larger than an additive effect” language of claim 38 merely characterizes an expected and inherent synergistic outcome of the co-expression system taught by Koprowski and does not confer patentable distinction.
Regarding Claim 39
Koprowski’s “green biomass” explicitly includes leaves and stems (¶[0029]) and the experimental examples are carried out on tobacco leaf tissue (¶¶[0118]–[0125]), including observations that TAG accumulation is increased in leaves. Thus, extracting and recovering lipid from transgenic leaves comprising DGAT and WR1/WRI1, as taught or suggested by Koprowski, would have been obvious.
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to TAM M NGUYEN whose telephone number is (571)272-1452. The examiner can normally be reached Mon - Frid.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Prem C Singh can be reached at 571-273-6381. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/TAM M NGUYEN/Primary Examiner, Art Unit 1771