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 .
For clarity, references to the instant specification will refer to the corresponding Pre-Grant Publication US 2023/0123431 A1.
Claim Status
The amended claim set filed on 13 August 2025 is acknowledged. Claims 1-21 are currently pending. Of those, claims 1, 4, and 13-14 are amended. There are no new claims and claims 15-16 are withdrawn. No claims are cancelled. Claims 1-14 and 17-21 will be examined on the merits herein.
Election/Restrictions
Applicant's election with traverse of Group I (claims 1-14) in the reply filed on 13 August 2025 (herein referred to as “Remarks”) is acknowledged. The traversal is on the ground(s) that claim 1 was amended to recite an isolated genetically engineered bacteria or yeast cell transformed to express genes encoding an octanoyltransferase, a lipoyl synthase, a protein substrate that is lipoylated, a lipoamidase and an S-adenosylmethionine synthase, rather than expressing at least one of the above genes as originally presented. Applicant argues (Remarks, pg. 6-7) that Liao and McFarlan (WO 02/085293) discloses the use of one of the above genes and does not teach or suggest the combination of all claimed lipoic acid pathway genes recited in claim 1. This argument is not found persuasive for Group II because claims 15-16 are drawn to a recombinant expression vector comprising one or more heterologous pathway genes defines in claim 1, i.e., the claims do not require all the limitations of the product of claim 1, and the technical feature of “one or more heterologous lipoic acid pathway genes defined in claim 1” (i.e., one or more of an octanoyltransferase, a lipoyl synthase, a lipoylated protein substrate, a lipoamidase, and S-adenosylmethionine synthase) still does not make a contribution over Liao and McFarlan. The requirement is still deemed proper and is therefore made FINAL.
Claims 15-16 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected invention, there being no allowable generic or linking claim. Applicant timely traversed the restriction (election) requirement in the reply filed on 13 August 2025.
Claims 17-21, directed to the invention(s) of a method of producing free lipoic acid in a genetically engineered cell, require all the limitations of the elected product claim of claim 1, are hereby rejoined and fully examined for patentability under 37 CFR 1.104, and claims 15-16 have NOT been rejoined.
Because a claimed invention previously withdrawn from consideration under 37 CFR 1.142 has been rejoined, the restriction requirement between groups I (claims 1-14) and III (claims 17-21) as set forth in the Office action mailed on 16 June 2025 is hereby withdrawn. In view of the withdrawal of the restriction requirement as to the rejoined inventions, applicant(s) are advised that if any claim presented in a divisional application is anticipated by, or includes all the limitations of, a claim that is allowable in the present application, such claim may be subject to provisional statutory and/or nonstatutory double patenting rejections over the claims of the instant application.
Once the restriction requirement is withdrawn, the provisions of 35 U.S.C. 121 are no longer applicable. See In re Ziegler, 443 F.2d 1211, 1215, 170 USPQ 129, 131-32 (CCPA 1971). See also MPEP § 804.01.
Priority
The instant application is a 371 of application PCT/SG2021/050103 (filed 2 March 2021) and claims priority to foreign application SG 10202001884X (filed 2 March 2020). Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55. Therefore, the effective filing date of instant claims 1-14 and 17-21 is 2 March 2020.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 2 September 2022 and 10 July 2025 are in compliance with the provisions of 37 CFR 1.97. Accordingly, each information disclosure statement is being considered by the examiner.
The listing of references in the specification is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Claim Objections
Claims 3 and 8 are objected to because of the following informalities: the genus names, “Kluyveromyces, Candida, Pichia, Yarrowia, Debaryomyces, Saccharomyces”, should be italicized. Appropriate correction is required.
Claim Interpretation
Claim 1 recites, “An isolated genetically engineered bacteria or yeast cell, wherein the cell has been transformed by at least one polynucleotide molecule… wherein… said genetically engineered bacteria or yeast cell is capable of increased production of free lipoic acid compared to a non-transformed cell.” As written, “a non-transformed cell” is not limited to a particular type of cell (such as the same species as the genetically engineered bacteria or yeast transformed by the at least one polynucleotide). Therefore, the broadest reasonable interpretation of this limitation is that the genetically engineered bacteria or yeast cell is capable of increased production of free lipoic acid compared to any non-transformed cell.
Claim Rejections - 35 USC § 112(b)
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 5-7 are 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 5 (upon which claims 6-7 depend) recites, “The genetically engineered bacteria or yeast cell of claim 1, wherein the lipoic acid pathway genes are expressed in mitochondria.” The term “expression” is used in the art to refer to various processes of protein synthesis (including transcription and translation), modification, and regulation (see “What is Protein Expression?”; PTO-892; para. 1). The instant specification does not specifically define the term “expression” alone or in the context of “express[ion] in mitochondria,” so it is unclear what expression process(es) are encompassed by the claim and occurring within mitochondria (i.e., are the genes incorporated into the mitochondrial genome and transcribed within the mitochondria, are the protein products encoded by the genes synthesized outside of the mitochondria and transported to the mitochondria or modified within the mitochondria, etc.), particularly because the claim states that “expression” is a process that happens to the genes rather than a protein encoded by the gene. Thus, one of ordinary skill in the art would not be able to determine how the limitation “the lipoic acid pathway genes are expressed in the mitochondria” limits the structure of the isolated genetically engineered cell of claim 1. Additionally, it is unclear how the claim limits the structure of an isolated genetically engineered bacterial cell of claim 1, as bacteria do not contain mitochondria. In the interest of compact prosecution, any step in the process of protein expression (e.g., transcription, translation, modification, etc.) within a mitochondria of a yeast cell is interpreted as meeting the limitation “lipoic acid pathway genes are expressed in mitochondria” as required in claims 5-7. It is suggested that claims 5-7 be limited to reciting yeast only. Clarification is requested.
Claim Rejections - 35 USC § 112(a)
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 1-14 and 17-21 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for the promoters and plasmids taught in the specification to result in particular expression levels of lipoamidase and lipoyl synthase and also being enabling for lipoic acid pathway protein localization in the mitochondria for yeast and not in the mitochondria for bacteria, does not reasonably provide enablement for any generic transformations of bacteria or yeast with lipoamidase or lipoyl synthase that lead to any possible expression level of those genes, and also does not reasonably provide enablement for yeast in which the lipoic acid pathway proteins are not targeted to the mitochondria or bacteria wherein the lipoic acid pathway proteins are targeted in mitochondria. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make the invention commensurate in scope with these claims.
The focus of the enablement inquiry is whether everything within the scope of the claim(s) is/are enabled, at the time of filing, without requiring undue experimentation to make or use the invention. The factors to be considered in determining whether a disclosure would require undue experimentation include: (A) The breadth of the claims; (B) The nature of the invention; (C) The state of the prior art; (D) The level of one of ordinary skill; (E) The level of predictability in the art; (F) The amount of direction provided by the inventor; (G) The existence of working examples; and (H) The quantity of experimentation needed to make or use the invention based on the content of the disclosure. In re Wands, 8 USPQ2d, 1400 (CAFC 1988) and MPEP 2164.01.
The breadth of the claims: With respect to claim breadth, the standard under 35 U.S.C. §112(a) or 35 U.S.C. §112, first paragraph, entails the determination of what the claims recite and what the claims mean as a whole. The claims are drawn to an isolated genetically engineered bacteria or yeast cell.
Claim 1, upon which claims 2-14 and 17-21 depend, recites, “An isolated genetically engineered bacteria or yeast cell, wherein the cell has been transformed by at least one polynucleotide molecule, the at least one polynucleotide molecule comprising lipoic acid pathway genes which encode an octanoyltransferase, a lipoyl synthase, a protein substrate that is lipoylated, a lipoamidase and an S-adenosylmethionine synthase, operably linked to at least one promoter, wherein the lipoic acid pathway genes are heterologous and said genetically engineered bacteria or yeast cell is capable of increased production of free lipoic acid compared to a non-transformed cell.”
The broadest reasonable interpretation of the claim is that the bacteria or yeast cell may be transformed with any plasmid lipoamidase and lipoyl synthase and using any promoter. Additionally, the functional limitation, “said genetically engineered bacteria or yeast cell is capable of increased production of free lipoic acid compared to a non-transformed cell”, requires that the cell having the polynucleotide molecule encoding the claimed genes be capable of producing free lipoic acid and producing more lipoic acid than some other cell.
Claim 5, upon which claims 6-7 depend, recites, “The isolated genetically engineered bacteria or yeast cell of claim 1, wherein the lipoic acid pathway genes are expressed in mitochondria.” The broadest reasonable interpretation of the claim is that the lipoic acid pathway genes are expressed in the mitochondria of bacteria or yeast.
Claims 17-21 are drawn to a method of producing free lipoic acid in a genetically engineered cell comprising the steps of culturing a plurality of genetically engineered cells of claim 1 in medium under conditions for lipoic acid biosynthesis and supplementing the medium with cysteine. Claims 17-19 do not limit the structure of the genetically engineered bacteria or yeast cell of claim 1. Claim 20, which depends upon claim 17, recites, “the engineered cell is a yeast cell.” Claim 21, which depends upon claim 20, recites, “the engineered cell is Saccharomyces cerevisiae.”
The nature of the invention; The nature of an isolated genetically engineered bacteria or yeast cell capable of producing free lipoic acid requires that the cell must be alive, the genes encoding the proteins of the lipoic acid pathway must be expressed, and proteins involved in the lipoic acid pathway must be functional and present in sufficient quantities to produce free lipoic acid.
The state of the prior art and the level of predictability in the art: Lennox-Hvenekilde et al. (2023, Metab Eng; herein “Lennox”) teaches that, as of the effective filing date of the claimed invention, there were few attempts at engineering or optimizing a lipoic acid cell factory in bacteria known in the art, and no attempts that produced free lipoic acid as required by the claims (pg. 41, left col., para. 1). MPEP 2124 states: “[R]eferences cited to show a universal fact need not be available as prior art before the effective filing date of applicant’s claimed invention. In re Wilson, 311 F.2d 266, 135 USPQ 442 (CCPA 1962)…. Some specific examples in which later publications showing factual evidence can be cited include situations where the facts shown in the reference are evidence "that, as of an application’s filing date, undue experimentation would have been required, In re Corneil, 347 F.2d 563, 568, 145 USPQ 702, 705 (CCPA 1965), or that a parameter absent from the claims was or was not critical, In re Rainer, 305 F.2d 505, 507 n.3, 134 USPQ 343, 345 n.3 (CCPA 1962)….” Lennox, though published after the effective filing date of the instant invention, summarizes teachings of the art prior to the effective filing date of the instant invention. Of the lipoic acid cell factories taught in Lennox, the work of Sun et al. (2017, PLoS ONE; cited in IDS) produced the most efficient production of lipoic acid (Lennox, pg. 41, left col., para. 1), which was done by cloning the lipD (lipoyl domain of E2 subunit), lipA (lipoyl synthase), and lplA (lipoate-protein ligase) genes in E. coli (Sun, abstract), but did not include a lipoamidase as in the instant claims and therefore did not produce free lipoic acid. Lennox also teaches that the art at the time of the effective filing date showed that the expression of LipA must be carefully balanced in order to properly function and that inclusion bodies form when expression levels are too high (pg. 43, right col., para. 3). None of the prior art teaches the expression of lipoamidase in bacteria for the purpose of lipoic acid production.
Reppas (US 2012/0164705 A1) teaches that lipoamidase can be toxic to host cells because of its high activity for lipoylated target substrates (para. 142), such as pyruvate dehydrogenase, which is crucial for aerobic respiration (Lennox, pg. 39, right col.). Spalding and Prigge (2009, PLoS ONE; cited in IDS) also teaches that lipoamidase (Lpa) is toxic to E. coli having a lipoic acid biosynthesis pathway because of its lipoamidase activity (Abstract and pg. 7, left col., para. 2). Lennox teaches that the art at the time of the effective filing date showed that because of this, the expression of Lpa must be sufficient to allow for optimal release of lipoic acid, but not so high as to halt the function of lipoic-acid-dependent enzymes (pg. 43, right col., para. 3). Similarly, Jiang and Cronan (2005, J Biol Chem; cited in IDS) teaches that the toxicity of Lpa precluded the use of high copy number vectors that are commonly used in E. coli expression systems and mandated the use of low copy number vectors (pg. 2249, left col., para. 2).
Thus, based on the teachings of the prior art, one of ordinary skill in the art would not be able to predict that any expression level of lipoamidase obtained by the use of any generic vector and/or any generic promoter would result in a genetically engineered bacteria or yeast cell capable of producing free lipoic acid, because the art shows that expression levels that are too high cause toxicity to the host cell.
Regarding the expression of the lipoic acid pathway in yeast, Schonauer et al. (2009, J Biol Chem) teaches that the substrate for de novo lipoic acid synthesis is octanoyl-ACP, which is synthesized by the mitochondrial type II synthetic pathway (FAS II), and that the lipoic acid pathway enzymes located in the mitochondria are Lip2 (octanoyltransferase), Lip5 (lipoyl synthase), Lip3 (homolog to lipoate-protein ligase), and Gcv3 (H protein of glycine cleavage enzyme) (pg. 23234, right col., para. 3 and pg. 23239, right col., para. 3). Thus, one of ordinary skill in the art would not be able to predict that an octanoyltransferase, lipoyl synthase, and Gcv3 could be expressed outside of the mitochondria in a yeast cell and result in a cell that is capable of increased production of free lipoic acid.
The amount of direction provided by the inventor and the existence of working examples: The instant specification teaches that lipoic acid synthesis and attachment in yeast is less well-understood than in bacteria, and the specification explicitly states that the activity of lipoamidase in yeast is unknown (para. 4). The specification does not reduce the invention to practice because there are no examples where all genes are heterologous. Instead, the instant specification teaches the production of lipoic acid by metabolic engineering of yeast, specifically by overexpressing endogenous Lip2, Lip5, Gcv3, and Sam1 or Sam2 genes from yeast and heterologously expressing Lpa from Enterococcus faecalis (para. 77 and Table 1). The specification teaches the use of only two plasmids, pIS385 and pRS41K, and four promoters, PGAL1, PTEF1, PPGI1, and PADH1 (para. 50 and Table 1). The specification teaches that EfLPA was expressed under the strong inducible promoter PGAL1 (para. 68) and that LIP5 (lipoyl synthase) must be expressed under a weak promoter because expression under a strong promoter caused cell inviability (i.e., the cells are not capable of increased production of lipoic acid).
Regarding the expression of the lipoic acid pathway in yeast, the specification teaches that lipoic acid synthesis occurs in the mitochondria of yeast (para. 71). “To enable lipoic acid biosynthesis in vivo, EfLPA must be translocated to the mitochondria where it hydrolyzes lipoic acid from lipoylated protein substrates.” (para. 71).
Therefore, what is enabled by the instant specification and working examples is narrow in comparison to the scope of the claims, and the specification does not provide enough information with which one may overcome the known unpredictability in the art.
The quantity of experimentation needed to make or use the invention based on the content of the disclosure: The standard of an enabling disclosure is not the ability to make and test if the invention works but one of the ability to make and use with a reasonable expectation of success. A patent is granted for a completed invention, not the general suggestion of an idea (MPEP 2164.03 and Chiron Corp. v. Genentech Inc., 363 F.3d 1247, 1254, 70 USPQ2d 1321, 1325-26 (Fed. Cir. 2004)). The instant specification is not enabling across the full scope claimed because one cannot follow the guidance presented therein, or within the art at the time of filing, and make or use the claimed product/composition or perform the claimed method without first making a substantial inventive contribution.
The instant specification and the teachings of the art prior to the effective filing date of the claimed invention both teach that certain expression levels of lipoamidase and lipoyl synthase are toxic. The art also teaches that lipoamidase has not previously been expressed with the other claimed enzymes in order to produce free lipoic acid. Therefore, one of ordinary skill in the art would need to determine what expression levels of lipoamidase and lipoyl synthase would not be toxic to the host cell and still increase production of free lipoic acid. This would require determining the expression levels for both the lipoamidase and the lipoyl synthase using different plasmids and promoters, followed by testing those vectors with the vector(s) encoding the remaining lipoic acid pathway genes to ensure enhanced free lipoic acid production. To do so without teachings from the prior art or the instant specification commensurate in scope with what is claimed would go beyond what is considered “routine” in the art.
The instant specification teaches that lipoic acid pathway proteins must be targeted to the mitochondria in yeast, and the art does not provide evidence that increased free lipoic acid production is possible by expressing lipoic acid pathway genes that are not targeted to the mitochondria. One would need to determine what, if any, lipoic acid pathway genes could be used to produce free lipoic acid outside of the mitochondria in yeast. To do so without some teaching from the prior art or the instant specification would go beyond what is considered “routine” in the art. Also, because bacteria do not have mitochondria, one cannot make a bacteria cell capable of increased lipoic acid production wherein the lipoic acid pathway genes are expressed in mitochondria.
Therefore, claims 1-14 and 17-21 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, for failing to meet the enablement requirement.
Claims 1-14 and 17-21 are 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. This is a written description rejection.
MPEP 2163.II.3.a.ii. states (emphasis added):
The written description requirement for a claimed genus may be satisfied through sufficient description of a representative number of species by actual reduction to practice (see i)(A) above), reduction to drawings (see i)(B) above), or by disclosure of relevant, identifying characteristics, i.e., structure or other physical and/or chemical properties, by 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 inventor was in possession of the claimed genus (see i)(C) above)…. A "representative number of species" means that the species which are adequately described are representative of the entire genus.
The broadest reasonable interpretation of claims 1 and 5 and the teachings of the art and the instant specification are set forth in the enablement rejection above (see para. 21-32).
Additionally, claim 1 recites, “wherein the lipoic acid pathway genes are heterologous”, meaning that all recited lipoic acid pathway genes encoded by the at least one polynucleotide must be heterologous. However, the specification only reduces to practice two Saccharomyces cerevisiae strains, both of which are transformed with only one heterologous gene, Enterococcus faecalis lipoamidase (EfLPA), and the other genes are S. cerevisiae genes, LIP2, LIP5, GCV3, and SAM1 or SAM2 (Table 1 and para. 50-51). The instant specification does not teach examples of lipoic acid genes from other bacteria or yeast species or indicate whether/how the expression system (e.g., vectors, promoters, targeting sequences, etc.) may vary depending on the genes used from different species and their expression in different host species.
The instant specification and the teachings of the art prior to the effective filing date of the claimed invention both teach that certain expression levels of lipoamidase and lipoyl synthase are toxic. The art also teaches that lipoamidase has not previously been expressed with the other claimed enzymes in order to produce free lipoic acid. Therefore, the species of promoters and plasmids described in the specification are not representative of the full genus of all possible promoters and plasmids due to the different expression levels having different results on the cell: either killing the cell, increasing the production of free lipoic acid, or being expressed in insufficient amounts to have an effect on the cell. Also, the specification does not establish a structure-function correlation that the claimed effect of increasing the production of free lipoic acid is correlated with all possible expression levels, as recited in the instant claims. Therefore, one of ordinary skill in the art would have concluded that the specification has not demonstrated possession of the full scope of plasmids, promoters, and expression levels claimed but instead has only demonstrated possession of an invention using the promoters and plasmids taught in the specification to result in particular expression levels of lipoamidase and lipoyl synthase.
Additionally, the instant specification and the teachings of the prior art prior to the effective filing date of the claimed invention both teach that in yeast, lipoic acid pathway proteins must be localized to the mitochondria and, in bacteria, lipoic acid pathway genes cannot be localized to mitochondria because bacteria do not contain mitochondria. The full scope of genetically engineered yeast cells claimed encompasses yeast cells in which lipoic acid pathway genes are not localized to mitochondria; thus, the species of yeast cells wherein lipoic acid pathway proteins are localized to the mitochondria described in the instant specification are not representative of the full scope of yeast cells claimed. Therefore, one of ordinary skill in the art would have concluded that the specification has not demonstrated possession of the full scope of genetically engineered bacteria or yeast cells claimed, but instead has only demonstrated possession of yeast cells in which lipoic acid pathway proteins are localized to the mitochondria and bacteria in which the lipoic acid pathway proteins are not localized to mitochondria.
The teachings of the art prior to the effective filing date of the claimed invention also teach that heterologous octanoyltransferase, lipoyl synthase, lipoylated protein substrate, and/or S-adenosylmethionine synthase have not previously been expressed in bacteria or yeast in order to produce free lipoic acid, and the instant specification only adequately describes two strains of genetically engineered yeast capable of producing free lipoic acid, both of which contain only one heterologous gene, lipoamidase. The species of genetically engineered bacteria or yeast cells described in the specification are not representative of the full scope claimed because the specification does not adequately describe a bacteria or yeast cell transformed with heterologous octanoyltransferase, lipoyl synthase, lipoylated protein substrate, and/or S-adenosylmethionine synthase. Therefore, one of ordinary skill in the art would have concluded that the specification has not demonstrated possession of a genetically engineered bacteria or yeast cell transformed with at least one polynucleotide encoding the claimed lipoic acid pathway genes, wherein all lipoic acid pathway genes are heterologous.
Therefore, claims 1-14 and 17-21 are rejected because the specification fails to demonstrate possession of a representative species within the claimed genus.
Conclusion
No claim is allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to BAILEY M MORGAN whose telephone number is (703)756-5388. The examiner can normally be reached M-F 9-5 ET.
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/BAILEY M MORGAN/Examiner, Art Unit 1645
/VANESSA L. FORD/Supervisory Patent Examiner, Art Unit 1674