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 .
Claim Status
Claims 1, 3-5, 7-9, 11-14, 16-24, 26-29, 31-33, 35-39 and 41-46 are pending and examined. Claims 2, 6, 10, 15, 25, 30, 34 and 40 have been cancelled.
Election/Restrictions
Applicant elected with traverse the species of a diterpene synthase, SEQ ID NO: 33 and ABS in the reply filed on 06 December 2022.
The requirement was deemed proper and made FINAL for the reasons as set forth in the Office action dated 08 February 2023.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 19, 21-24, 26, 27, 29, 31-33, 35, 36, 38, 39, 41 and 43-46 REMAIN rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 19 is drawn to a method comprising cultivating cells comprising at least one expression vector comprising a first nucleic acid segment encoding a first lipid droplet surface protein (LDSP).
Meanwhile, the specification discloses that an LDSP or a portion thereof can increase manufacture of various terpenoid products, that the LDSP or a portion thereof can be linked in frame with a fusion partner such as a terpene synthase, and that one type of lipid droplet associated protein is a lipid droplet surface protein (p. 11).
Here, the metes and bounds of the claim are indefinite because LDSP is not an art-recognized term, because the specification fails to specifically define LDSP, because the only example of an LDSP in the art is SEQ ID NO: 1, and because the specification describes LDSP by function rather than structure such that one would not know what is or is not encompassed by the limitation LDSP.
Claims 21, 22, 23, 33, 36, 38 and 39 recite the same limitation and are therefore rejected for the same reason as provided for claim 19.
Claims 24, 26, 27, 29, 31, 32, 35, 41 and 43-46 are rejected for depending upon a rejected base claim and for failing to remedy the issues of indefiniteness.
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim 5 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 5 is drawn to an expression systems requiring a cytochrome P450 and an LDSP having as little as 95% sequence identity to SEQ ID NO: 1 and wherein the LDSP has a sequence with at least 90% sequence identity to SEQ ID NO: 1.
Here, claim 5 fails to further limit the claim from which it depends because it is broader in scope by claiming that the LDSP has a sequence with at least 90% sequence identity to SEQ ID NO: 1.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1, 3-5, 7-9, 11-14, 16-24, 26-29, 31-33, 35-39 and 41-46 REMAIN rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Instant claims 1, 3-5, 7-9, 11-14, 16-24, 26-29, 31-33, 35-39 and 41-46 are drawn to compositions, expression systems, methods and hosts requiring a cytochrome P450 and an LDSP or an LDSP having as little as 90% or 95% sequence identity to SEQ ID NO: 1 or a truncated LDSP with as little as 95% sequence identity to a sequence consisting at as few as 70 contiguous amino acids of SEQ ID NO: 1 where the truncated sequence localizes the LDSP within or at the surface of lipid droplets (LDs).
The specification describes that an LDSP or a portion thereof can increase manufacture of various terpenoid products, that the LDSP or a portion thereof can be linked in frame with a fusion partner such as a terpene synthase, and that one type of lipid droplet associated protein is a lipid droplet surface protein (p. 11).
The specification describes the expression of various genes with full-length LDSP in plants produced various terpenes and triacylglycerol (see Examples beginning on p. 113; see Example 6).
The written description requirement may be satisfied through sufficient description of a representative number of species by disclosing relevant and identifying characteristics such as structural or other physical and/or chemical properties, by disclosing functional characteristics coupled with a known or disclosed correlation between function and structure, or by a combination of such identifying characteristics, sufficient to show the applicant was in possession of the invention as claimed. See Eli Lilly,119 F.3d at 1568, 43 USPQ2d at 1406.
In the instant matter, the specification has failed to, in fact, describe a representative number of LDSP species as broadly encompassed by claims 19, 21, 22, 33 and 38, has failed to describe an LDSP having a sequence with as little as 90% or 95% sequence identity to SEQ ID NO: 1 or a truncated LDSP with as little as 95% sequence identity to a sequence consisting at as few as 70 contiguous amino acids of SEQ ID NO: 1, has failed to describe a representative number of species from the broad genus of cytochrome P450s, and has failed to describe a representative number of hosts in the methods as claimed.
Here, a polypeptide with as little as 90% or 95% identity to SEQ ID NO: 1 would have 15 and 7 amino acid substitutions and would encompass 1915 and 197 distinct protein variants, respectively. However, in the absence of guidance indicating where in the sequence of SEQ ID NO: 1 such variations can be sustained, the skilled practitioner would not be of the opinion that Applicant possesses the genus of LDSP proteins that retain functional activity for use in the compositions, methods and hosts as claimed.
This description is also critical because aside from Vieler et al which suggests that LDSP may be used in a similar fashion to plant oleosins (2012, Plant Physiology, 158:1562-1569; p. 1567, last ¶), the art fails to describe the particular structures conferring LDSP functional activity. In fact, Applicant’s own arguments support this position: there are structural and functional differences between LDSP and oleosin (Appeal Brief dated 30 June 2025, p. 20, ¶ 1).
In other words, even though LDSP having the amino acid sequence of SEQ ID NO: 1 and oleosin could be used interchangeably, one cannot predictably determine the structures required for LDSP function based on the critical domains and motifs of oleosin as described in the art, and thus would be unable to use the broad genus of variant LDSP as encompassed by the claims.
The Federal Circuit has clarified the application of the written description requirement. The court stated that a written description of an invention "requires a precise definition, such as by structure, formula, [or] chemical name, of the claimed subject matter sufficient to distinguish it from other materials." University of California v. Eli Lilly and Co., 119 F.3d 1559, 1568; 43 USPQ2d 1398, 1406 (Fed. Cir. 1997).
The court also concluded that "naming a type of material generally known to exist, in the absence of knowledge as to what that material consists of, is not a description of that material." Id.
Further, the court held that to adequately describe a claimed genus, Patent Owner must describe a representative number of the species of the claimed genus, and that one of skill in the art should be able to "visualize or recognize the identity of the members of the genus." Id. See MPEP 2163.
Applicant should note that the written description requirement serves to warn an innocent purchaser of the infringement of a patent, and conversely requires the patentee to distinguish the invention in the disclosure, and thus prevents the inventor from practicing upon the credulity or fears of other persons or from pretending that the invention is more than what it is. see Vas-Cath Inc. v. Mahurkar 1991 (CA FC) 19 USPQ2d 1111, 1115.
Here, the instant specification has merely described that SEQ ID NO: 1 has LDSP functional activity but has failed to either describe a representative number of species with functional activity from the broad genus of LDSPs as claimed (e.g., see sequence search results), and has further failed to describe the structures conferring LDSP functional activity.
As such, these claims are “reach through” claims in which Applicant has only described a starting material and at least one method step, but has not described the resulting product such that the genus of products that can be produced by the recited method steps and materials is so large that one of skill in the art would not readily envision the members of the claimed genus. (See Univ. of Rochester v. G.D. Searle & Co., 358 F.3d 916, 920-23, 69 USPQ2d 1886, 1890-93 (Fed. Cir. 2004)).
With respect to a “cytochrome P450” as claimed, the specification has failed to describe a representative number of species from the broad genus of P450s that retain functional activity.
This description is critical as the art describes that cytochrome P450s constitute the largest and functionally most versatile enzyme superfamily in nature having extremely diverse amino acid sequence participating in many different biological processes (Pateraki et al, 2015, Adv. Biochem Eng Biotechnol, 148:107-139, p. 111, ¶ 1).
Moreover, creative and efficient approaches are emerging to identify the fraction of genes relevant for terpenoid biosynthesis, particularly in regard to the identification of P450s as these exist in high numbers in plant genomes (p. 113, last ¶).
Here, the specification fails to describe how to identify cytochrome P450s as claimed that possess the functional activity required to participate in the synthesis of terpenoids, and fails to distinguish between those that have the required function and those which do not.
Therefore, in light of the breadth of the claims, the failure of the specification to describe the critical domains or motifs required for LDSP functional activity, the state of the art and the absence of a structure-function relationship for LDSP in the literature, and the failure to provide a representative number of species from the broad genus of cytochrome P450s as claimed the skilled artisan would not be of the opinion that Applicant possesses the compositions, expression systems, hosts and methods as broadly claimed.
Response to Arguments
Applicant traverses the rejection of the claims because the genetic code has been deciphered for at least 50 years and because with knowledge of the genetic code and Applicant's disclosure to guide them, those of ordinary skill in the art are fully capable of writing down any nucleic or amino acid sequence having 95% homology or higher to another sequence.
Applicant continues that in view of the disclosure of SEQ ID NO: 1, those skilled in the art could readily envision all of the amino acid sequences that are 95% identical to SEQ ID NO: 1. Such manipulations of nucleic acids, amino acids and testing are routine to those of ordinary skill in the art as indicated by the USPTO Written Description Training Materials, March 25, 2008, Example 10 (Applicant reply dated 26 March 2026, p. 11, ¶ 1).
This argument is not persuasive because the example to which Applicant refers indicates the written description may be satisfied for claims directed to structures and variants thereof when no function is claimed. Here, a function is claimed for the genus of structures. In this scenario, Example 10 of the USPTO Written Description Training Materials, March 25, 2008 concludes the specification does not provide an adequate written description.
Moreover, these arguments do not address whether a truncated sequence consisting of at least 70 contiguous amino acids of SEQ ID NO: 1 retain functional activity to localize the lipid droplet surface protein within or at the surface of lipid droplets.
Finally, Applicant’s arguments are not commensurate in scope with what is claimed as claims 19, 21, 22, 33 and 38 fail to recite any particular structure for the LDSP.
Applicant argues that the specification provides guidance on the functions of the protein structure of LDSP and that there is literal support for LDSP having the claimed percent identity (Applicant reply dated 23 March 2026, p. 12).
This argument is not persuasive because Applicant’s arguments are not commensurate in scope with what is claimed: claims 19, 21, 22, 33 and 38 fail to recite any particular structure for the LDSP. Moreover, the specification fails to describe any structure/function correlation shared among the genus of amino acid sequences as claimed.
Applicant’s arguments also appear to be contradictory. On the one hand Applicant argues that the skilled artisan would think Applicant possesses the genus of variants as claimed due to the similarity between oleosins and LDSP because oleosin function is known and each are lipid droplet associated proteins but on the other hand argues that one would not have a reasonable expectation of success in substituting oleosins for LDSP because they are structural and functional differences between the two (e.g., see Applicant reply dated 23 March 2026, p. 17).
Regarding CYPs, Applicant argues the written description requirement has been satisfied because two examples of CYPs have been provided (Applicant reply 23 March 2026, p. 13, part B.).
This argument is not persuasive because the specification has failed to describe a representative number of species from the broad genus of P450s that retain functional activity.
This description is critical as the art describes that cytochrome P450s constitute the largest and functionally most versatile enzyme superfamily in nature having extremely diverse amino acid sequence participating in many different biological processes (e.g., see Pateraki, p. 111, ¶ 1).
Moreover, creative and efficient approaches are emerging to identify the fraction of genes relevant for terpenoid biosynthesis, particularly in regard to the identification of P450s as these exist in high numbers in plant genomes (p. 113, last ¶).
Here, the specification fails to describe how to identify cytochrome P450s as claimed that possess the functional activity required to participate in the synthesis of terpenoids, and fails to distinguish between those that have the required function and those which do not.
Claim Rejections - 35 USC § 103
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.
Claim(s) 1, 4, 5, 7, 11, 12, 14, 16-22, 26, 27, 29, 31, 32, 33, 35, 36, 38, 39, 41 and 43-46 REMAIN rejected under 35 U.S.C. 103 as being unpatentable over Yuan et al (Pub. No. US 2017/0226526 A1) and Kinney et al (Patent No. US 7,256,014 B2) and Pateraki et al (2015, Adv. Biochem Eng Biotechnol, 148:107-139 citing Dai et al, 2014, Scientific Reports, 4:1-6), in view of Vieler et al (2012, Plant Physiology, 158:1562-1569).
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 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.
Instant claims 1, 4, 5, 7, 11, 12, 14, 16-22, 26, 27, 29, 31, 32, 33, 35, 36, 38, 39, 41 and 43-46 are drawn to a composition comprising a first fusion protein comprising a LDSP having the amino acid sequence of SEQ ID NO: 1 and a second fusion protein comprising a LDSP linked in-frame to a cytochrome P450, an expression system comprising said fusion protein, wherein the plastid targeting signal is removed or a hydrophobic region is linked in frame with the diterpene synthase and/or cytochrome P450, wherein the nucleic acid encoding said proteins are codon optimized, a Nicotiana plant comprising said expression system, a method of incubating said host cell and isolating lipids therefrom wherein the system comprises two expression cassettes, and a fusion protein comprising a LDSP having the amino acid sequence of SEQ ID NO: 1 linked in frame to a terpene synthase that produces a lipophilic product that localizes to lipids droplets in a cytosol of a host cell wherein the product is a terpenoid and where the host has at least 300 micrograms terpenoids per gram fresh weight and wherein the terpenoid is monoterpene, diterpene or sesquiterpene.
Yuan et al claim a genetic construct comprising a promoter and an oleosin peptide that leads to the formation of a droplet body comprising a biological product and an enzyme including FPPS, SQS or both to increase production of the product or of terpene production, wherein the construct comprises a signal peptide, wherein the enzyme may also be GPPS, a recombinant vector comprising said genetic construct, a method for increasing yield of a terpenoid or terpene in a plant comprising transforming a plant, and products obtained by said methods (see claims 1, 2, 4, 6, 7, 10, 12-14, 23, 24, 30, 32, 33, 37, 42 and 49).
Yuan et al teach that said methods may be practiced in soybean or tobacco and that expression of said fusion protein forms droplet bodies in the chloroplast and cytosol and that the peptide comprises a hydrophobic region (p. 7, ¶ 0103; see also p. 5, ¶ 0087; see ¶ 0004).
Yuan et al teach that a successful design for the production of hydrophobic terpenes requires co-localization of terpene biosynthesis and droplet formation (¶ 0064 and ¶ 0075). Production of the desired product may be in any desired organelle or part of the plant cell where the compartmentalizing protein comprises both hydrophobic and hydrophilic regions (¶ 0068 and ¶ 0075-0076, ¶ 0084).
Yuan et al teaches using oleosin that is not targeted to the cytosol for the production of a protein of interest (see claims 1 and 2 versus claim 6; see also ¶ 0067, 0068 and 0076 and 0099), and that it was known that oleosins are involved in the cytosolic production of lipid droplets (p. 3, ¶ 0065; see also ¶ 0070).
To summarize, Yuan et al teaches a fusion protein comprising a first fusion partner comprising a diterpene synthase and an oleosin to produce terpenes in LDs within the cytosol of a host plant cell.
Similarly, Kinney et al claim a recombinant microbial host or method for the production of hydrophobic compounds comprising at least one genetic construct encoding an oleosin polypeptide wherein the compound is an isoprenoid and the host is algae, wherein the construct is operably linked to a constitutive or inducible promoter, and wherein the product is recovered (see claims 1, 2, 5, 7, 8, 9, 11, 12 and 14).
Kinney et al teach that GGDPS, DXS or FDPS can be expressed to convert pyruvate and G3P to FPP to yield monoterpenes, sesquiterpenes or diterpenes including tetraterpenoids such as carotenoids (col. 10, ¶ 2; see also col. 17, ¶ 3 and last ¶). Genes may be codon-optimized, and any promoter available to one of skill in the art may be used to express said genes (col. 12, last ¶; col. 14, last ¶).
The expression system is not limited to bacteria and is not limited to the production of carotenoids (col. 10, ¶ 2; see also col. 17, ¶ 3 and last ¶; see claims directed to the use blue/green algae Spirulina).
Meanwhile, Pateraki et al teaches it was known in the art that terpenoid biosynthesis involves two main classes of enzymes: terpenes synthases and cytochrome p450s (p. 110, last ¶).
The use of plant hosts for the production of terpenoids appears to be an obvious approach because precursors, cofactors and critical enzymes such as P450 are endogenously present (p. 126, ¶ 1). Terpenoids have, in fact, been produced in transgenic Nicotiana in substantial amounts (p. 126, ¶ 1).
Pateraki et al disclose that coexpression of both P450 and germacrene A synthase can lead to the production of transgenic sesquiterpenes (p. 126, last ¶). A problem to be solved is the storage of produced terpenoids in specialized structures (p. 127, last ¶). Thus it is clear that the art recognizes that diterpene synthases and cytochrome P450s should be expressed in the same compartmentalized location in the plant cell.
Pateraki et al teaches that plant systems are uniquely suited to high value terpenoid production and that it is expected that novel compounds can be produced by expressing enzymes from different terpenoid pathways and P450, with several groups having already demonstrated that this approach is feasible with novel triterpenes being generated from nonnatural combinations of plant-derived triterpene synthases and P450s expressed in yeast (p. 128; see also Dai et al).
Taken together, the prior art reasonably teaches, suggests and provides motivation for compositions, hosts and expression systems comprising a first fusion protein comprising an oleosin and diterpene synthase that is expressed and compartmentalized with a second cytochrome P450 protein in order to couple the proteins in the same location which are required to produce terpenoids having various commercial applications.
Thus, the issue is whether one would have modified the first fusion protein as taught by Yuan et al by instead using a LDSP protein having the amino acid sequence of SEQ ID NO: 1 as found in N. oceanica in addition to modifying the cytochrome P450 as taught by Pateraki et al to make a second fusion protein comprising said P450 and an LDSP that is coexpressed/compartmentalized with said first fusion protein in a plant to produce a desired terpenoid.
To this point, Vieler et al teach that oleosins are known in the art to be employed in molecular farming allowing for targeting to the lipid droplets or the endoplasmic reticulum (p. 1567, col. 1, last ¶).
Vieler et al teach that the particularly claimed LDSP was known in the art as GenBank Accession No. JQ268559 and having 100% sequence identity to SEQ ID NO: 1 of the instant invention, and which displayed similar function to an Arabidopsis oleosin (see Attachment A in the Office action dated 08 February 2023; see also p. 1568, col. 1, ¶ 1; see p. 1564, col. 1, last ¶).
Vieler teaches in the last paragraph of page 1563 that oleosins and LDSP share structural features:
Similar to oleosins, LDSP of Nannochloropsis possesses an uninterrupted sequence of 62 amino acids with nonpolar side chains. Even through this is shorter than the hydrophobic region of oleosins, it theoretically allows the formation of a hairpin or similar structure sufficiently long to reach beyond the phospholipid monolayer surrounding TAGs.
Vieler et al specifically teaches in the last paragraph of page 1567:
LDSP might be used in biotechnological applications similar to those conceived for oleosins (Capuano et al., 2007). Oleosins are employed in molecular farming, allowing the targeting of proteins or peptides to LDs or the endoplasmic reticulum (Pons et al., 2009), and have been proposed to stabilize emulsions and artificial LDs. The localization of LDSP to LDs in plants suggests that it might localize to any LD, delivering a given protein to a LD due to its inherent hydrophobic characteristics. Thus, LDSP might provide an additional reagent with diverging properties in the tool box of bioengineers.
Therefore, prior to the effective filing date of the instant invention, it would have been prima facie obvious to one of ordinary skill in the art to modify the teachings of Yuan et al and Kinney et al and Pateraki et al where diterpene synthases and cytochrome p450s are coexpressed and compartmentalized by instead using an LDSP for example, as taught by Vieler et al because oleosins and LDSPs are functionally equivalent and would lead to the each of the enzymes required for terpene production being compartmentalized in the same location: a lipid droplet.
Namely, one would have found it obvious to do so because the amino acid sequence of SEQ ID NO: 1 having LDSP activity is functionally interchangeable with the proteins and oleosins as taught by Yuan et al and Kinney et al and Pateraki et al and are used for the same purpose of compartmentalizing proteins of interest to the lipid droplet where both the diterpene synthase and cytochrome P450 can simultaneously produce terpenoids.
One would have found it obvious to either remove a plastid targeting sequence from the diterpene synthase and the cytochrome P450, or in the alternative use a hydrophobic region linked in frame with the diterpene synthase, because it would ensure the compartmentalization of enzymes in the same structure to synthesize the desired product as Vieler et al teaches the localization of LDSP to LDs in plants suggests that it might localize to any LD, delivering a given protein to a LD due to its inherent hydrophobic characteristics.
One would have a reasonable expectation of success in doing so because similar to oleosins, LDSP possesses an uninterrupted sequence of 62 amino acids with nonpolar side chains theoretically allowing the formation of a hairpin or similar structure sufficiently long to reach beyond the phospholipid monolayer surrounding TAGs, and because Vieler et al specifically posits that LDSP might be used in biotechnological applications similar to those conceived for oleosins.
Response to Arguments
Applicant traverses the rejection of the claims because Yuan, Kinney and Pateraki do not teach any fusion protein comprising LDSP and a diterpene synthase or LDSP and cytochrome P450, and that said references are silent on any advantages of said fusion proteins (Applicant reply dated 23 March 2026, p. 15, section 1).
This argument is not persuasive because arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
Here, Applicant merely argues what is or is not taught by the references rather than explaining why would not have modified the cited references in light of the teachings of Vieler.
Regarding the argument that the references fail to teach “fusion proteins”, it is noted that Yuan claims a genetic construct comprising a promoter and compartmenting peptide and which further comprises an enhancing protein to enhance the production of a biological product wherein the enhancing protein is a terpene synthase (see claims 1, 7 and 12). Thus it is clear that Yuan does, in fact, teach a first fusion protein as claimed (see also ¶ 0123).
Applicant argues there is no reasonable expectation of success in arriving at the claims based on the cited references because oleosins and LDSP are not functionally interchangeable based on various teachings in Vieler and because there are structural and functional differences between LDSP and oleosin (Applicant reply dated 23 March 2026, p. 16, section 2).
However, this argument is unpersuasive because one would have a reasonable expectation of success in substituting oleosin for LDSP because similar to oleosins, LDSP possesses an uninterrupted sequence of 62 amino acids with nonpolar side chains theoretically allowing the formation of a hairpin or similar structure sufficiently long to reach beyond the phospholipid monolayer surrounding TAGs (e.g., see Vieler. p. 1563 as cited supra), and because Vieler et al specifically posits that LDSP might be used in biotechnological applications similar to those conceived for oleosins.
Applicant’s arguments also appear to be contradictory. On the one hand Applicant argues that one would not have a reasonable expectation of success in substituting oleosins for LDSP because there are structural and functional differences between the two and on the other hand argues that the skilled artisan would think Applicant possesses the genus of variants as claimed due to the similarity between oleosins and LDSP because oleosin function is known and each are lipid droplet associated proteins (e.g., see Applicant reply dated 23 March 2026, p. 12).
Applicant asserts that the Yuan teaches that only the hydrophobic core of oleosin can be used requiring the removal of hydrophilic regions and thus would be contrary to the explicit teachings of Yuan if LDSP is substituted for oleosin (Applicant reply dated 23 March 2026, p. 17, last ¶).
Applicant’s argument is not persuasive because the teachings of Yuan are not, in fact, contrary to that of Vieler: the purpose of both Yuan and Vieler is for molecular pharming to produce a protein/product of interest in a lipid droplet.
In any event, Yuan teaches the compartmenting peptide (i.e., the oleosin) may comprise both hydrophobic and hydrophilic regions ( ¶0084).
Applicant argues the Office has failed to provide sufficient objective motivation because there is no need to change or improve the teachings of Yuan or Kinney (Applicant reply dated 26 March 2026, p. 18, ¶ 1).
This argument is unpersuasive because the basis of the rejection is not that one needs to improve upon the teachings of Yuan or Kinney. Rather, a prima facie case of obviousness has been presented pursuant to MPEP 2143(I)(B) where one would simply substitute one known element (i.e., oleosins) for another (i.e., LDSP) to obtain predictable results.
Claim(s) 1, 3, 4, 8, 13, 19, 23, 28, 33, 37, 38 and 42 REMAIN rejected under 35 U.S.C. 103 as being unpatentable over Yuan et al (Pub. No. US 2017/0226526 A1) and Kinney et al (Patent No. US 7,256,014 B2) and Pateraki et al (2015, Adv. Biochem Eng Biotechnol, 148:107-139 citing Dai et al, 2014, Scientific Reports, 4:1-6), and in view of Vieler et al (2012, Plant Physiology, 158:1562-1569) and Chappell et al (Patent No. US 10,597,665 B1, referred to herein as ‘665).
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 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.
Instant claims 1, 3, 4, 13, 19, 23, 28, 33, 37, 38 and 42 are drawn to compositions, hosts, expression systems and methods comprising a first fusion protein comprising a diterpene synthase and an LDSP or an LDSP having the amino acid sequence of SEQ ID NO: 1 and a second fusion protein comprising a LDSP linked in-frame to a cytochrome P450, wherein the diterpene synthase comprises a polypeptide having the amino acid sequence of SEQ ID NO: 33 which is an ABS enzyme.
Yuan et al claim a genetic construct comprising a promoter and an oleosin peptide that leads to the formation of a droplet body comprising a biological product and an enzyme including FPPS, SQS or both to increase production of the product or of terpene production, wherein the construct comprises a signal peptide, wherein the enzyme may also be GPPS, a recombinant vector comprising said genetic construct, a method for increasing yield of a terpenoid or terpene in a plant comprising transforming a plant, and products obtained by said methods (see claims 1, 2, 4, 6, 7, 10, 12-14, 23, 24, 30, 32, 33, 37, 42 and 49).
Yuan et al teach that said methods may be practiced in soybean or tobacco and that expression of said fusion protein forms droplet bodies in the chloroplast and cytosol and that the peptide comprises a hydrophobic region (p. 7, ¶ 0103; see also p. 5, ¶ 0087; see ¶ 0004). A successful design for the production of hydrophobic terpenes requires co-localization of terpene biosynthesis and droplet formation (¶ 0064 and ¶ 0075). Production of the desired product may be in any desired organelle or part of the plant cell where the compartmentalizing protein comprises both hydrophobic and hydrophilic regions (¶ 0068 and ¶ 0075-0076, ¶ 0084).
Yuan et al teaches using oleosin that is not targeted to the cytosol for the production of a protein of interest (see claims 1 and 2 versus claim 6; see also ¶ 0067, 0068 and 0076 and 0099), and that it was known that oleosins are involved in the cytosolic production of lipid droplets (p. 3, ¶ 0065; see also ¶ 0070).
To summarize, Yuan et al teaches a fusion protein comprising a first fusion partner comprising a diterpene synthase and an oleosin to produce terpenes in LDs within the cytosol of a host plant cell.
Similarly, Kinney et al claim a recombinant microbial host or method for the production of hydrophobic compounds comprising at least one genetic construct encoding an oleosin polypeptide wherein the compound is an isoprenoid and the host is algae, wherein the construct is operably linked to a constitutive or inducible promoter, and wherein the product is recovered (see claims 1, 2, 5, 7, 8, 9, 11, 12 and 14).
Kinney et al teach that GGDPS, DXS or FDPS can be expressed to convert pyruvate and G3P to FPP to yield monoterpenes, sesquiterpenes or diterpenes including tetraterpenoids such as carotenoids (col. 10, ¶ 2; see also col. 17, ¶ 3 and last ¶). Genes may be codon-optimized, and any promoter available to one of skill in the art may be used to express said genes (col. 12, last ¶; col. 14, last ¶).
The expression system is not limited to bacteria and is not limited to the production of carotenoids (col. 10, ¶ 2; see also col. 17, ¶ 3 and last ¶; see claims directed to the use blue/green algae Spirulina).
Meanwhile, Pateraki et al teaches it was known in the art that terpenoid biosynthesis involves two main classes of enzymes: terpenes synthases and cytochrome p450s (p. 110, last ¶).
The use of plant hosts for the production of terpenoids appears to be an obvious approach because precursors, cofactors and critical enzymes such as P450 are endogenously present (p. 126, ¶ 1). Terpenoids have, in fact, been produced in transgenic Nicotiana in substantial amounts (p. 126, ¶ 1).
Pateraki et al disclose that coexpression of both P450 and germacrene A synthase can lead to the production of transgenic sesquiterpenes (p. 126, last ¶). A problem to be solved is the storage of produced terpenoids in specialized structures (p. 127, last ¶). Thus it is clear that the art recognizes that diterpene synthases and cytochrome P450s should be expressed in the same compartmentalized location in the plant cell.
Pateraki et al teaches that plant systems are uniquely suited to high value terpenoid production and that it is expected that novel compounds can be produced by expressing enzymes from different terpenoid pathways and P450, with several groups have already demonstrated that this approach is feasible with novel triterpenes being generated from nonnatural combinations of plant-derived triterpene synthases and P450s expressed in yeast (p. 128; see also Dai et al).
Taken together, the prior art reasonably teaches, suggests and provides motivation for compositions, hosts and expression systems comprising a first fusion protein comprising an oleosin and diterpene synthase that is expressed and compartmentalized with a second cytochrome P450 protein in order to ensure the proteins which are required to produce terpenoids having various commercial applications are in the same location in the cell.
Thus, the issue is whether one would have modified the first fusion protein as taught by Yuan et al by instead using a LDSP protein having the amino acid sequence of SEQ ID NO: 1 as found in N. oceanica in addition to modifying the cytochrome P450 as taught by Pateraki et al to make a second fusion protein comprising said P450 and an LDSP that is coexpressed/compartmentalized with said first fusion protein in a plant to produce a desired terpenoid, and whether one would have further expressed the diterpene synthase ABS protein having the amino acids sequence of SEQ ID NO: 33.
To this point, Vieler et al teach that oleosins are known in the art to be employed in molecular farming allowing for targeting to the lipid droplets or the endoplasmic reticulum (p. 1567, col. 1, last ¶).
Vieler et al teach that the particularly claimed LDSP was known in the art as GenBank Accession No. JQ268559 and having 100% sequence identity to SEQ ID NO: 1 of the instant invention, and which displayed similar function to an Arabidopsis oleosin (see Attachment A in the Office action dated 08 February 2023; see also p. 1568, col. 1, ¶ 1; see p. 1564, col. 1, last ¶).
Vieler teaches in the last paragraph of page 1563 that oleosins and LDSP share structural features:
Similar to oleosins, LDSP of Nannochloropsis possesses an uninterrupted sequence of 62 amino acids with nonpolar side chains. Even through this is shorter than the hydrophobic region of oleosins, it theoretically allows the formation of a hairpin or similar structure sufficiently long to reach beyond the phospholipid monolayer surrounding TAGs.
Vieler et al specifically teaches in the last paragraph of page 1567:
LDSP might be used in biotechnological applications similar to those conceived for oleosins (Capuano et al., 2007). Oleosins are employed in molecular farming, allowing the targeting of proteins or peptides to LDs or the endoplasmic reticulum (Pons et al., 2009), and have been proposed to stabilize emulsions and artificial LDs. The localization of LDSP to LDs in plants suggests that it might localize to any LD, delivering a given protein to a LD due to its inherent hydrophobic characteristics. Thus, LDSP might provide an additional reagent with diverging properties in the tool box of bioengineers.
Meanwhile, ‘665 teaches that the diterpene abietadiene is known to have various commercial applications (see Table 1), and that the ABS protein producing said diterpene is designated as SEQ ID NO: 18 and has 100% sequence identity to SEQ ID NO: 33 of the instant invention (see Attachment A in the Office action dated 08 February 2023). ‘665 teaches, in fact, expression of ABS alone or in conjunction with GGPP synthase yields abietadiene (see Fig. 6 and 7; see also col. 8).
Therefore, prior to the effective filing date of the instant invention, it would have been prima facie obvious to one of ordinary skill in the art to modify the teachings of Yuan et al and Kinney et al and Pateraki et al where diterpene synthases and cytochrome p450s are coexpressed and compartmentalized in the same location by instead using an LDSP instead of an oleosin for example, as taught by Vieler et al, because oleosins and LDSPs are functionally equivalent.
Namely, one would have found it obvious to do so because the amino acid sequence of SEQ ID NO: 1 having LDSP activity is functionally interchangeable with the proteins and oleosin as taught by Yuan et al and Kinney et al and Pateraki et al and are used for the same purpose of targeting proteins of interest to the lipid droplet for the compartmentalized production of terpenoids.
One would have a reasonable expectation of success in doing so because similar to oleosins, LDSP possesses an uninterrupted sequence of 62 amino acids with nonpolar side chains theoretically allowing the formation of a hairpin or similar structure sufficiently long to reach beyond the phospholipid monolayer surrounding TAGs, and because Vieler et al specifically posits that LDSP might be used in biotechnological applications similar to those conceived for oleosins.
One would have found it obvious to, and with a reasonable expectation of success, to also use an expression vector to express a nucleic acid encoding an ABS protein having the amino acid sequence of SEQ ID NO: 33 or wherein said amino acid sequence is comprised in the first fusion partner or the first nucleic acid segment because said polypeptide was known in the art and leads to the predictable production of abietadiene, a terpenoid whose importance is noted above.
Claim(s) 4, 9, 19 and 24 REMAIN rejected under 35 U.S.C. 103 as being unpatentable over Yuan et al (Pub. No. US 2017/0226526 A1) and Kinney et al (Patent No. US 7,256,014 B2) and Pateraki et al (2015, Adv. Biochem Eng Biotechnol, 148:107-139 citing Dai et al, 2014, Scientific Reports, 4:1-6) and in view of Vieler et al (2012, Plant Physiology, 158:1562-1569) Delatte et al (2018, Plant Biotechnology Journal, 16:1997-2006 published on 28 May 2018).
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 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.
Instant claims 4, 9, 19 and 24 are drawn to an expression system and method comprising a first fusion protein comprising a diterpene synthase and an LDSP and a second fusion protein comprising a LDSP linked in-frame to a cytochrome P450, wherein the system or methods further comprises an expression cassette comprising a promoter operably linked to a nucleic acid sequence encoding a WRI1 transcription factor.
Yuan et al claim a genetic construct comprising a promoter and an oleosin peptide that leads to the formation of a droplet body comprising a biological product and an enzyme including FPPS, SQS or both to increase production of the product or of terpene production, wherein the construct comprises a signal peptide, wherein the enzyme may also be GPPS, a recombinant vector comprising said genetic construct, a method for increasing yield of a terpenoid or terpene in a plant comprising transforming a plant, and products obtained by said methods (see claims 1, 2, 4, 6, 7, 10, 12-14, 23, 24, 30, 32, 33, 37, 42 and 49).
Yuan et al teach that said methods may be practiced in soybean or tobacco and that expression of said fusion protein forms droplet bodies in the chloroplast and cytosol and that the peptide comprises a hydrophobic region (p. 7, ¶ 0103; see also p. 5, ¶ 0087; see ¶ 0004). A successful design for the production of hydrophobic terpenes requires co-localization of terpene biosynthesis and droplet formation (¶ 0064 and ¶ 0075). Production of the desired product may be in any desired organelle or part of the plant cell where the compartmentalizing protein comprises both hydrophobic and hydrophilic regions (¶ 0068 and ¶ 0075-0076, ¶ 0084).
Yuan et al teaches using oleosin that is not targeted to the cytosol for the production of a protein of interest (see claims 1 and 2 versus claim 6; see also ¶ 0067, 0068 and 0076 and 0099), and that it was known that oleosins are involved in the cytosolic production of lipid droplets (p. 3, ¶ 0065; see also ¶ 0070).
To summarize, Yuan et al teaches a fusion protein comprising a first fusion partner comprising a diterpene synthase and an oleosin to produce terpenes in LDs within the cytosol of a host plant cell.
Similarly, Kinney et al claim a recombinant microbial host or method for the production of hydrophobic compounds comprising at least one genetic construct encoding an oleosin polypeptide wherein the compound is an isoprenoid and the host is algae, wherein the construct is operably linked to a constitutive or inducible promoter, and wherein the product is recovered (see claims 1, 2, 5, 7, 8, 9, 11, 12 and 14).
Kinney et al teach that GGDPS, DXS or FDPS can be expressed to convert pyruvate and G3P to FPP to yield monoterpenes, sesquiterpenes or diterpenes including tetraterpenoids such as carotenoids (col. 10, ¶ 2; see also col. 17, ¶ 3 and last ¶). Genes may be codon-optimized, and any promoter available to one of skill in the art may be used to express said genes (col. 12, last ¶; col. 14, last ¶).
The expression system is not limited to bacteria and is not limited to the production of carotenoids (col. 10, ¶ 2; see also col. 17, ¶ 3 and last ¶; see claims directed to the use blue/green algae Spirulina).
Meanwhile, Pateraki et al teaches it was known in the art that terpenoid biosynthesis involves two main classes of enzymes: terpenes synthases and cytochrome p450s (p. 110, last ¶).
The use of plant hosts for the production of terpenoids appears to be an obvious approach because precursors, cofactors and critical enzymes such as P450 are endogenously present (p. 126, ¶ 1). Terpenoids have, in fact, been produced in transgenic Nicotiana in substantial amounts (p. 126, ¶ 1).
Pateraki et al disclose that coexpression of both P450 and germacrene A synthase can lead to the production of transgenic sesquiterpenes (p. 126, last ¶). A problem to be solved is the storage of produced terpenoids in specialized structures (p. 127, last ¶). Thus it is clear that the art recognizes that diterpene synthases and cytochrome P450s should be expressed in the same compartmentalized location in the plant cell.
Pateraki et al teaches that plant systems are uniquely suited to high value terpenoid production and that it is expected that novel compounds can be produced by expressing enzymes from different terpenoid pathways and P450, with several groups have already demonstrated that this approach is feasible with novel triterpenes being generated from nonnatural combinations of plant-derived triterpene synthases and P450s expressed in yeast (p. 128; see also Dai et al).
Taken together, the prior art reasonably teaches, suggests and provides motivation for compositions, hosts and expression systems comprising a first fusion protein comprising an oleosin and diterpene synthase that is expressed and compartmentalized with a cytochrome P450 protein, both of which are required to produce terpenoids having various commercial applications and ensures the proteins are in the same location.
Thus, the issue is whether one would have modified the first fusion protein as taught by Yuan et al by instead using a LDSP protein having the amino acid sequence of SEQ ID NO: 1 as found in N. oceanica in addition to modifying the cytochrome P450 as taught by Pateraki et al to make a second fusion protein comprising said P450 and an LDSP that is coexpressed/compartmentalized with said first fusion protein in a plant to produce a desired terpenoid, and whether one would have expressed said proteins in addition to a WRI1 transcription factor.
To this point, Vieler et al teach that oleosins are known in the art to be employed in molecular farming allowing for targeting to the lipid droplets or the endoplasmic reticulum (p. 1567, col. 1, last ¶).
Vieler et al teach that the particularly claimed LDSP was known in the art as GenBank Accession No. JQ268559 and having 100% sequence identity to SEQ ID NO: 1 of the instant invention, and which displayed similar function to an Arabidopsis oleosin (see Attachment A in the Office action dated 08 February 2023; see also p. 1568, col. 1, ¶ 1; see p. 1564, col. 1, last ¶).
Vieler teaches in the last paragraph of page 1563 that oleosins and LDSP share structural features:
Similar to oleosins, LDSP of Nannochloropsis possesses an uninterrupted sequence of 62 amino acids with nonpolar side chains. Even through this is shorter than the hydrophobic region of oleosins, it theoretically allows the formation of a hairpin or similar structure sufficiently long to reach beyond the phospholipid monolayer surrounding TAGs.
Vieler et al specifically teaches in the last paragraph of page 1567:
LDSP might be used in biotechnological applications similar to those conceived for oleosins (Capuano et al., 2007). Oleosins are employed in molecular farming, allowing the targeting of proteins or peptides to LDs or the endoplasmic reticulum (Pons et al., 2009), and have been proposed to stabilize emulsions and artificial LDs. The localization of LDSP to LDs in plants suggests that it might localize to any LD, delivering a given protein to a LD due to its inherent hydrophobic characteristics. Thus, LDSP might provide an additional reagent with diverging properties in the tool box of bioengineers.
Delatte et al teach, in fact, increasing storage capacity for sesquiterpenes by coexpressing diacylglycerol acyltransferases, WRI1 and oleosins (see Abstract; see also p. 1998, col. 2; see p. 1999, col. 2).
Therefore, prior to the effective filing date of the instant invention, it would have been prima facie obvious to one of ordinary skill in the art to modify the teachings of Yuan et al and Kinney et al and Pateraki et al where diterpene synthases and cytochrome p450s are coexpressed by instead using an LDSP for example, as taught by Vieler et al because oleosins and LDSPs are functionally equivalent.
Namely, one would have found it obvious to do so because LDSP activity is functionally interchangeable with the oleosin and proteins as taught by Yuan et al and Kinney et al and Pateraki et al and are used for the same purpose of targeting proteins of interest to the lipid droplet for the compartmentalized production of terpenoids.
One would have a reasonable expectation of success in doing so because similar to oleosins, LDSP possesses an uninterrupted sequence of 62 amino acids with nonpolar side chains theoretically allowing the formation of a hairpin or similar structure sufficiently long to reach beyond the phospholipid monolayer surrounding TAGs, and because Vieler et al specifically posits that LDSP might be used in biotechnological applications similar to those conceived for oleosins.
One would have found it obvious, and with a reasonable expectation of success, to also express an expression cassette comprising a promoter operably linked to a nucleic acid encoding a WRI1 transcription factor because Dalette et al teach, in fact, increasing storage capacity for sesquiterpenes by expressing WRI1 and oleosin, the latter of which would be substituted with LDSP for the reasons as set forth supra.
Conclusion
No claim is allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/JASON DEVEAU ROSEN/Primary Examiner, Art Unit 1662
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