DETAILED ACTION
Notice of Pre-AIA or AIA Status
1. The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
2. Please note that the Patent Examiner of your application has changed. All communications should be directed to Mary Lyons, Art Unit 1645, whose telephone number is (571)272-2966.
Claim Status
3. The amendment, filed 09/26/25, has been entered.
4. Claims 1-3, 5, 13, 16-19, 21, 24, 32-35 and 38 are pending. Claims 4, 6-12, 14-15, 20, 22-23, 25-31, and 36-37 are cancelled. Claims 1-3, 5, 13, 19, 21, 24, 32, and 33 are amended. Claims 19, 21, 24, 32-35 and 38 have been withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 12/09/24. Claims 1-3, 5, 13, and 16-18 are under examination.
Declaration under 37 C.F.R. 1.132
5. The declaration under 37 CFR 1.132 filed 09/26/25 is insufficient to overcome the rejection of claims 1-7, 13, and 16-18 based upon 35 U.S.C. 112(a) as failing to comply with the written description requirement or the rejection of claims 1-7, 13, and 16-18 based upon 35 U.S.C. 112(a) for lack of enablement, as set forth in the last Office action because when all of the evidence is considered, the totality of the rebuttal evidence of adequate written description and enablement fails to outweigh the evidence of inadequate written description and lack of enablement, especially with regards to newly added limitations encompassing the use of partial structures (e.g. as little as 70% sequence variations); see maintained rejections below. Thus, there is no showing that the objective evidence of adequate written description or enablement is commensurate in scope with the claims, as written.
Withdrawal of Objections/Rejections
6. The following are withdrawn from the Office Action, filed 03/28/25:
The objection to the drawings, found on page 3, is withdrawn in light of Applicant’s submission of replacement drawings.
The objection to claim 7, found on page 4, is moot in light of Applicant’s cancellation thereof.
The rejection of claims 1-6 under 35 U.S.C. 112(b) as being indefinite, found on page 4, is withdrawn in light of Applicant’s amendments thereto (or moot in light of Applicant’s cancellation thereof).
The rejection of claims 3-6 under 35 U.S.C. 112(d) as being of improper dependent form, found on page 16, is withdrawn in light of Applicant’s amendments thereto (or moot in light of Applicant’s cancellation thereof).
The rejections of claims 1-7, 13, 16, and 18 under 35 U.S.C. 102(a)(1) as being anticipated by Anderson (AU Application No. 2008200780B2) is withdrawn in light of Applicant’s amendments thereto (or moot in light of Applicant’s cancellation thereof).
The rejection of claims 1-7, 13, and 16-18 under 35 U.S.C 103 as being unpatentable over Anderson (AU Application No. 2008200780B2), found on page 21, is withdrawn in light of Applicant’s amendments thereto (or moot in light of Applicant’s cancellation thereof).
Maintained Rejection: Claim Rejections - 35 USC § 112
7. 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.
8. Claims 1-3, 5, 13, and 16-18 are rejected under 35 U.S.C. 112(a) 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, at the time the application was filed, had possession of the claimed invention.
The claims, even as amended, broadly encompass a large genus of recombinant heterologous gene sequences defined as, inter alia, those encoding heterologous enzymes including wherein the enzyme is N-monooxygenase and the N-monooxygenase consists of an amino acid sequence at least 70% identical to the amino acid sequence of SEQ ID NO: 7. Accordingly, the claim scope is vast and highly varied based on the sequence variants encompassed. However, the specification does not provide sufficient written description to inform a skilled artisan which domains or residues can differ from those of SEQ ID NO: 7, while still retaining functional activity, and thus does not provide evidence that the Applicant had full possession of the claimed invention. Claim 1 is representative of the pending claims scope and broadly recites “A recombinant cell for producing para-nitro-L-phenylalanine (pN-Phe), comprising heterologous genes encoding heterologous enzymes, expressing the heterologous enzymes and native chorismate and converting the chorismate to pN-Phe using the heterologous enzymes, wherein the heterologous enzymes comprise PapA, PapB, PapC and a N-monooxygenase, wherein the N-monooxygenase consists of an amino acid sequence at least 70% identical to the amino acid sequence of SEQ ID NO: 7, and wherein the recombinant cell is Escherichia coli.
However, it is the Office’s position that the sequence variants having at least 70% identity to SEQ ID NO: 7 have not been described with sufficient particularity, such that one skilled in the art would recognize that Applicant had possession of the claimed invention, at the time of filing, because of (A) a lack of a correlation, known or disclosed, between the claimed functional requirements and the structures that meet those requirements; and/or (B) a lack of a representative number and variety of species to constitute possession of the full scope of the claimed genus.
For example, the specification does not provide adequate written description to identify the broad genus of the claims because, inter alia, the specification does not disclose a correlation between the necessary structure of the claimed polypeptide functional homologue (e.g. which 70% of the amino acids must be maintained and which 30% may be substituted within a N-monooxygenase sequence variant prior to back translation into a polynucleotide (i.e. a heterologous gene) to be incorporated into the E. coli host cell) and the claimed function to be maintained (e.g. an E. coli cell that produces para-nitro-L-phenylalanine; pN-Phe). It is noted that while the description of the ability of a claimed protein sequence may generically describe that protein molecule's function, it does not describe the protein molecule itself, and thus, by extension cannot describe the polynucleotide gene sequence that encodes it. For example, the specification fails to identify critical amino acids or subsequences within SEQ ID NO: 7 that must be retained in order to maintain the claimed functional activity. Consequently, the specification fails to describe the common attributes or structural characteristics that identify the members of this genus and because the genus of sequences is highly variable (i.e. each sequence has a unique structure; see MPEP 2434), the characteristics of the ability to encode a poorly described partial structure (i.e. an enzyme with less than 100% similarity), is insufficient to describe the genus. Thus, the specification does not provide substantive evidence for possession of this large and variable genus, encompassing a massive number of partial structures claimed only by a functional characteristic because, without an art-recognized structure-function correlation, the capability to recognize or understand the structure from the mere recitation of function and minimal structure is highly unlikely. Thus, disclosure of function alone is little more than a wish for possession and it does not satisfy the written description requirement; See Eli Lilly, 119 F.3d at 1568, 43 USPQ2d at 1406 (written description requirement not satisfied by merely providing "a result that one might achieve if one made that invention"); In re Wilder, 736 F.2d 1516, 1521, 222 USPQ 369, 372-73 (Fed. Cir. 1984) (affirming a rejection for lack of written description because the specification does "little more than outline goals appellants hope the claimed invention achieves and the problems the invention will hopefully ameliorate").
Further, MPEP §2163 states that if a biomolecule is described only by a functional characteristic (as in the instant case), without any disclosed correlation between function and structure of the sequence (as in the instant case), it is not sufficient for written description purposes, even when accompanied by a method of obtaining the claimed sequences. MPEP §2163 does state that for a generic claim the genus can be adequately described if the disclosure presents a sufficient number of representative species that encompass the genus. If the genus has a substantial variance, the disclosure must describe a sufficient variety of species to reflect the variation within that genus. Although the MPEP does not define what constitutes a sufficient number of representative species, the courts have indicated what does not constitute a representative number to adequately describe a broad genus. For example, the courts determined that the disclosure of two chemical compounds within a subgenus did not describe that subgenus (e.g. see In re Gostelli, 872, F.2d at 1012, 10 USPQ2d at 1618). Furthermore, the disclosure of only one or two species encompassed within a genus adequately describes a claim directed to that genus only if the disclosure "indicates that the patentee has invented species sufficient to constitute the gen[us]. "See Enzo Biochem, 323 F.3d at 966, 63 USPQ2d at 1615; Noelle v. Lederman, 355 F.3d 1343, 1350, 69 USPQ2d 1508, 1514 (Fed. Cir. 2004) (Fed. Cir. 2004) "[A] patentee of a biotechnological invention cannot necessarily claim a genus after only describing a limited number of species because there may be unpredictability in the results obtained from species other than those specifically enumerated."). "A patentee will not be deemed to have invented species sufficient to constitute the genus by virtue of having disclosed a single species when ... the evidence indicates ordinary artisans could not predict the operability in the invention of any species other than the one disclosed." In re Curtis, 354 F.3d 1347, 1358, 69 USPQ2d 1274, 1282 (Fed. Cir. 2004).
In the instant case, the specification provides complete structural information for SEQ ID NO: 7 (i.e. a polypeptide sequence for a N-monooxygenase). However, the claims, as written, also encompass the use of partial structures of SEQ ID NO: 7, subsequently back-translated into polynucleotides gene sequences which are to then be incorporated into E. coli for the production of pN-Phe. More specifically, SEQ ID NO: 7 consists of 339 amino acids. Therefore, a sequence variant having at least 70% identity to SEQ ID NO: 7 means that up to and including 101 changes (i.e. 1 to 101 amino acid substitutions) in any combination of positions is within the claim scope (i.e. 238/339*100 = 70%; 339-238 = 101). However, there are an almost unfathomable number of ways in which 1 to 101 amino acids (i.e. the sum of results for 1 change, 2 changes, 3 changes … 101 changes) can be selected from the 339 residues because, without any other limitations in the independent claim, each particular residue can be substituted with any one of the other 19 naturally occurring amino acids and still meet the limitation. For example, for just 1 change (i.e. only one amino acid substitution within SEQ ID NO: 7), there would be 339 unique sequences (i.e. each with only one difference), but because the change at each residue can be substituted with any one of the other 19 naturally occurring amino acids, then the actual number of unique sequences encompassed is 6,441 (i.e. a sequence having a substitution at, for example, residue 33 with a cysteine, would be structurally distinct from a sequence having a substitution at residue 33 with a tryptophan; 6,441 = 339 * 19). Yet, the claims allow for up to 101 unrestricted substitutions, anywhere along the length of SEQ ID NO: 7, so with order of selection not important and repetition not allowed, the equation is X = 19 * [n!/(r!(n-r)!)], which - for 101 changes, results in a number having more than 250 zeros (i.e. a trillion only has 12 zeros). Each of which would then need to be back translated into a gene sequence to be incorporated into the E. coli host cell for the production of pN-Phe). However, the instant specification only provides working examples related to the engineered biosynthetic pathway using recombinant E. Coli (MG1655) with the heterologous papABC genes and ObaC. The recombinant E. Coli (MG1655) was further modified via the overexpression of the AroG protein which catalyzes the first step in chorismate synthesis (Specification, page 7). Therefore, the specification also does not provide adequate written description to identify the broad genus of the claims because, inter alia, the specification does not disclose a sufficient number and variety of species.
In the absence of a reduction to practice of a representative number and variety of species, the written description requirement for a claimed genus may be satisfied by disclosure of relevant, identifying characteristics, sufficient to show the applicant was in possession of the claimed genus. Yet, the specification only provides working examples using recombinant E. coli MG1655 and Pn-Phe is a non-standard amino acid and as noted by the instant specification, “Although pN-Phe can be incorporated site-specifically within proteins in live cells, there is currently no means to biosynthesize pN-Phe within live cells” (Specification, page 1). Therefore, in the absence of working examples, the degree of predictability with regards to the necessary heterologous gene sequences encoding heterologous enzyme sequences is relatively low. There is a high degree of complexity and variability in the biosynthetic pathways and metabolism within the exemplary cell types listed in the specification. This includes significant differences with E. coli MG1655 regarding the function of enzymes within biosynthetic pathways, which is critical to the ability of the invention to comprise the conversion of metabolites to products within the recombinant cells.
Thus, the breadth of the claim scope is vast regarding heterologous genes that the invention can comprise. The specification discusses heterologous genes from Streptomyces venezuelae (papABC) or Pseudomonas fluorescens (obaCDE), and includes information regarding the scope of sequence similarity of the pap genes to those in Streptomyces venezuelae when the native metabolite is chorismate (Specification, Page 19). Regarding aminotransferases and N-oxygenases capable of performing the claimed conversions to pN-Phe, the specification notes that the most probable native aminotransferase in E. coli that catalyzes this reaction is TyrB but it is likely that multiple aminotransferases can contribute to the formation of the amino acid” (i.e. the state of the art is under development, and thus necessarily unpredictable; see Specification, Page 7). Further, the Figure 2 description states, “the stability of the heterologous metabolites of the pathway in the presence of metabolically active E. coli in culture media including pA- Phe, pA-Pyr, pN-Phe, and pN-Pyr. The stability of the desired product pN-Phe is an important criterion to determine for success of this invention. Our results indicate that phenylalanine derivatives are fairly stable, whereas pyruvate derivatives are comparatively unstable. The latter instability may be due to endogenous aminotransferase activity.” (Specification, page 7). Thus, either there are endogenous aminotransferases that the inventors do not provide description for, pyruvate derivatives are unstable for other unknown reasons, or both. In order to satisfy the written description requirement for heterologous enzymes, as broadly claimed, the claims require the artisan to envisage all genes encoding all enzymes that are capable of performing the claimed conversion; however, the specification itself is uncertain which enzymes accomplish this goal.
With regards to the state of the art and related to the present invention Butler et al., in “De novo biosynthesis of para-nitro-L-phenylalanine in Escherichia coli”, 2021, bioRxiv, pgs., 1-40, states, “Despite exploration of several N-oxygenases …as potential in vitro catalysts for nitroaromatic product synthesis, thus far, no N-oxygenases have been used in vivo for de novo biosynthetic cascades despite their promise and demonstrated activity in E. coli” (Page 5, lines 105-110). The Butler further notes, “high accumulation of pA-Phe could result in off-target nsAA incorporation if a strain were to perform biosynthesis and site-specific nsAA incorporation…Thus, to improve product titer and make pN-Phe the dominant nsAA product, we sought to identify an N-oxygenase enzyme with improved activity on pA-Phe… ObaC is the only diiron monooxygenase thus far to exhibit full oxidation of pA-Phe to pN-Phe…To test a diverse set of N-oxygenase enzymes, we selected twenty-one distinct sequences from 18 different clusters to obtain diversity in the diiron monooxygenase sequence space from those currently characterized and ordered gene fragments… Fermentation with metabolically active cells for 24 h at 30 °C revealed that only one of the additional N-oxygenases resulted in pN-Phe production apart from ObaC. This N-oxygenase was NO16 from Pseudomonas sp. EMN2, which exhibited greater than twice the pN-Phe production in the initial screen” (Table 1; Pages 21-24, lines 423-464).
Furthermore, given the highly diverse nature of nucleic acid molecules, even one of skill in this art cannot envision the structure of a corresponding nucleotide sequence by only knowing what it encodes, as evidenced by the art, for example, see Edelman et al. 2001 (Degeneracy and complexity in biological systems; PNAS 98(24): 13763-13768). In summary, a specific protein sequence is easily determined from a known nucleic acid sequence, because the known sequence of nucleotides (i.e. a nucleic acid sequence) produces one and only one corresponding protein sequence (i.e. a sequence of amino acids); but, the inverse is not true because a specific sequence of nucleotides cannot be ascertained from a known sequence of amino acids since more than one distinct codon (i.e. set of three nucleotides) may code for a particular amino acid. Thus, a description of the amino acid per se, does not provide sufficient written description of the codon encoding that amino acid because the one-to-one correspondence is unidirectional, i.e., from codon forward to amino acid, but not from amino acid back-translated to codon. Thus, the specification does not adequately describe any of the structures of the numerous nucleic acid sequences claimed by their capacity to encode the claimed protein sequence, especially a poorly describe partial structure thereof. Thus, given the vast number of recombinant heterologous gene sequences (i.e. more than a billion trillion, see math above) that fall within the scope of the claims, there is a lack of written description regarding suitable heterologous gene sequences encoding poorly described partial structures (i.e. 70% sequence variants) of the heterologous enzymes capable for use in the invention.
Although the level of skill in the art is high, the predictability in the art is low due to the complexity of biological systems, de novo engineering biosynthetic pathways, enzymology, and vast differences in metabolism that fall within the scope of the invention. Specifically, an artisan would not be able to predict or identify, a priori, and in the absence of any guidance or consensus structures exactly what heterologous gene sequences would be suitable for the invention and accordingly, an artisan would not reasonably conclude that Applicant possessed the full scope of the broad and varied claim scope.
Consequently, neither the specification nor the state of the art provides sufficient written description to support the genus encompassed by the claims. Vas-Cath Inc. v. Mahurkar, 19 USPQ2d 1111, makes clear that "applicant must convey with reasonable clarity to those skilled in the art that, as of the filing date sought, he or she was in possession of the invention. The invention is, for purposes of the 'written description' inquiry, whatever is now claimed." (See page 1117.) The specification does not "clearly allow persons of ordinary skill in the art to recognize that [he or she] invented what is claimed." (See Vas-Cath at page 1116.). Given the above analysis of the factors as a whole, which the courts have determined are critical in determining whether Applicant is in possession of or the specification supports the claimed invention, Applicant has not satisfied the requirements as set forth under 35 U.S.C. 112(a).
Applicant’s Arguments and Response to Arguments
9. All of Applicant’s arguments have been considered but were not deemed persuasive; accordingly, the rejection is maintained for reasons of record. For example:
With regards to the argument that the specification provides adequate description of the recombinant cell of claim 1, as amended, in, for example, pages 7-11, 18, 20-23, and 25-36, FIGS. 1-16, and the original claims (see Remarks, page 9); the Office disagrees and notes a careful review of these citations does not provide adequate support for the sequence variants having at least 70% identity to SEQ ID NO: 7. Thus this argument is not persuasive because it remains the Office’s position that these variants have not been described with sufficient particularity, such that one skilled in the art would recognize that Applicant had possession of the claimed invention, at the time of filing, because of (A) a lack of a correlation, known or disclosed, between the claimed functional requirements and the structures that meet those requirements; and/or (B) a lack of a representative number and variety of species to constitute possession of the full scope of the claimed genus.
With regards to most of the arguments based on the declaration (see Remarks, pages 9-11 and Declaration paragraphs 3, 4, 5); the Office disagrees and notes none of these citations addresses support for the new limitations pertaining to the use of partial structures of SEQ ID NO 7 each of which needs to be back translated into a heterologous gene for incorporation into an E. coli strain. Thus, these arguments are not persuasive because they are on not on point with the issue raised.
With regards to the declaration at paragraphs 6 and 8-11 (see Remarks, at page 10), it is noted that even if SEQ ID NO: 16 and 22-29 (i.e. 9 sequences) represented species within the genus of partial structures represented by the newly added limitation “… wherein the enzyme is N-monooxygenase and the N-monooxygenase consists of an amino acid sequence at least 70% identical to the amino acid sequence of SEQ ID NO: 7” it remains the Office’s position that these 9 sequences do not constitute both a representative number and variety for the billions of trillions (i.e. well over 1 x 10250 partial structures of enzymes that must also then be back translated to an unfathomable number of gene sequences). Accordingly, this argument is not persuasive because it is not commensurate in scope with the scope of the claims.
Therefore, all of Applicant’s arguments have been considered but were not deemed persuasive; accordingly, the rejection is maintained for reasons of record because when all of the evidence is considered, the totality of the rebuttal evidence of adequate written description fails to outweigh the evidence of inadequate written description, especially with regards to newly added limitations encompassing the use of an unimaginable number of partial structures (e.g. as little as 70% sequence variations).
Maintained Rejection: Claim Rejections - 35 USC § 112
10. Claims 1-3, 5, 13, and 16-18 are rejected under 35 U.S.C. 112(a) as failing to comply with the enablement requirement. The claim contains subject matter which was not described in the specification in such a way as to enable one skilled in the art to which it pertains, or with which it is most nearly connected, to make and/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. Although all the factors were considered, the most relevant ones are discussed below.
Nature of the invention: The nature of the invention is recombinant cells for producing para-nitro-L-phenylalanine (pN-Phe), comprising heterologous genes encoding heterologous enzymes, expressing the heterologous enzymes and native chorismate and converting the chorismate to pN-Phe using the heterologous enzymes, wherein the heterologous enzymes comprise PapA, PapB, PapC and a N-monooxygenase, wherein the N-monooxygenase consists of an amino acid sequence at least 70% identical to the amino acid sequence of SEQ ID NO: 7, and wherein the recombinant cell is Escherichia coli. Therefore, the nature of the invention is a chemical case, where there is natural unpredictability in performance of certain species or sub-combinations other than those specifically enumerated; see MPEP 2163. Accordingly, it is the Office’s position that undue experimentation would be required to practice the claimed invention, with a reasonable expectation of success, because it would not be predictable from the disclosure of one particular species (e.g. a fully described SEQ ID NO: 7 in E. coli host cells) what other species (e.g. other sequence variations back translated and incorporated therein) may or may not work; see MPEP 2164.03.
Breadth of the claims: With respect to claim breadth, the standard under 35 U.S.C. §112(a), entails the determination of what the claims recite and what the claims mean as a whole. In addition, when analyzing enablement, the claims are analyzed with respect to the teachings of the specification and are to be “given their broadest reasonable interpretation consistent with the specification.” See MPEP 2111 [R-5]; Phillips v. AWH Corp., 415 F.3d 1303, 75 USPQ2d 1321 (Fed. Cir. 2005); and In re Hyatt, 211 F.3d 1367, 1372, 54 USPQ2d 1664, 1667 (Fed. Cir. 2000). Applicant always has the opportunity to amend the claims during prosecution, and broad interpretation by the examiner reduces the possibility that the claim, once issued, will be interpreted more broadly than is justified. In re Prater, 415 F.2d 1393, 1404-05, 162 USPQ 541, 550- 51 (CCPA 1969). The broadest reasonable interpretation of the instant claims covers numerous partial structures (i.e. sequence variations) subsequently back translated and incorporated into E. coli host cells. However, without guidance on which of the structural components are required (i.e. which amino acids must be conserved in a variant, and then which nucleic acids encode for them) to maintain their claimed functions (i.e. produce pN-Phe) and without a disclosed correlation between function and structure, undue experimentation would be required to determine which of the numerous structures actually work. Accordingly, undue experimentation would be required to practice the claimed invention, with a reasonable expectation of success, because while enablement is not precluded by the necessity for routine screening, if a large amount of screening is required, the specification must provide a reasonable amount of guidance with respect to the direction in which the experimentation should proceed and such guidance has not been provided in the instant specification (see below).
Amount of direction provided by Inventor and Existence of Working Examples: The specification discloses, “Although pN-Phe can be incorporated site-specifically within proteins in live cells, there is currently no means to biosynthesize pN-Phe within live cells” (i.e. the art is under development and thus, necessarily unpredictable; see Specification, page 1). Therefore, in the absence of working examples, the degree of predictability with regards to the necessary heterologous gene sequences encoding heterologous enzyme sequences is relatively low. There is a high degree of complexity and variability in the biosynthetic pathways and metabolism within the exemplary cell types listed in the specification. This includes significant differences with E. coli MG1655 regarding the function of enzymes within biosynthetic pathways, which is critical to the ability of the invention to comprise the conversion of metabolites to products within the recombinant cells. The specification only provides working examples using recombinant E. coli MG1655 and pN-Phe is a non-standard amino acid and as noted by the instant specification. Further, the inventors find that pyruvate derivatives are less stable than phenylalanine derivatives, which indicates that either there are endogenous aminotransferases that the application is not enabled for, the pyruvate derivatives are unstable for other unknown reasons, or both. In either case it would take undue experimentation to test for the stability of pyruvate derivatives, and find unknown aminotransferases that might be performing the biosynthetic conversions. In order to satisfy the enablement requirement for the N-oxygenase enzymes, as broadly claimed, the claims require the artisan to predict how the few disclosed enzymes relate to the broadly claimed genus.
While the specification provides experimental detail regarding use of the engineered biosynthetic pathway for the conversion of native metabolites and heterologous enzymes to pN-Phe, and describes the enzymatic steps by which the conversions likely take place, the claims are not enabled for the conversion of metabolites to pN-Phe in an engineered system other than recombinant E. Coli exogenously expressing the papABC genes. The specification notes that there are uncertainties related to the aminotransferases that perform the biosynthetic conversions, as well as stability differences between critical metabolite derivatives. The specification lacks sufficient guidelines and working examples that would enable the use of recombinant cell types other than E. Coli exogenously expressing the papABC genes, to perform the claimed biosynthetic conversions.
Accordingly, the scope of the claims is extremely broad compared to the guidance and exemplification provided in the specification and the specification fails to teach the necessary structure-function correlations and/or a representative number and variety of species, as set forth above. Therefore, the only way to determine if the functional property of a sequence variant is indeed retained, is empirical testing of each and every variant back translated into a heterologous gene sequence for use in a strain of E. coli. Consequently, based on the almost unfathomable number of possibilities, a non-routine amount of experimentation would be required to practice the invention, with a reasonable expectation of success, because testing such a vast number of options would be easily recognized by the skilled practitioner to be disproportionately demanding and thus rise to the level of non-routine.
State of the Prior Art and Level of Predictability in the Art: With regards to the state of the art and related to the present invention Butler et al., in “De novo biosynthesis of para-nitro-L-phenylalanine in Escherichia coli”, 2021, bioRxiv, pgs., 1-40, states, “Despite exploration of several N-oxygenases …as potential in vitro catalysts for nitroaromatic product synthesis, thus far, no N-oxygenases have been used in vivo for de novo biosynthetic cascades despite their promise and demonstrated activity in E. coli” (Page 5, lines 105-110). The Butler further notes, “high accumulation of pA-Phe could result in off-target nsAA incorporation if a strain were to perform biosynthesis and site-specific nsAA incorporation…Thus, to improve product titer and make pN-Phe the dominant nsAA product, we sought to identify an N-oxygenase enzyme with improved activity on pA-Phe… ObaC is the only diiron monooxygenase thus far to exhibit full oxidation of pA-Phe to pN-Phe…To test a diverse set of N-oxygenase enzymes, we selected twenty-one distinct sequences from 18 different clusters to obtain diversity in the diiron monooxygenase sequence space from those currently characterized and ordered gene fragments… Fermentation with metabolically active cells for 24 h at 30 °C revealed that only one of the additional N-oxygenases resulted in pN-Phe production apart from ObaC. This N-oxygenase was NO16 from Pseudomonas sp. EMN2, which exhibited greater than twice the pN-Phe production in the initial screen” (Table 1; Pages 21-24, lines 423-464). Therefore, as discussed for the aminotransferases above, the claims require the artisan to envisage all enzymes that are capable of performing the claimed conversions; however, the specification itself only describes the use of ObaC and sequences that share the ObaC enzymatic activity.
Thus, Butler et al. disclose attempts to improve pA-Phe generation for the in vivo de novo biosynthetic cascades by screening for suitable N-oxygenase enzymes. The authors tested a diverse range of N-oxygenase enzymes, but only identified one additional suitable enzyme besides ObaC. Further, the literature discloses only two N-oxygenases capable of performing the necessary pN-Phe conversion. Given the high degree of difficulty and unpredictability of experimentation (metabolic and enzymatic assays) combined with the large variability between cell types means that a skilled artisan would incur an undue amount of experimentation to engineer the biosynthetic system for each different cell type, heterologous enzyme, and cell culture condition.
Furthermore, given the highly diverse nature of nucleic acid molecules, even one of skill in this art cannot envision the structure of a corresponding nucleotide sequence by only knowing what it encodes, as evidenced by the art, for example, see Edelman et al. 2001 (Degeneracy and complexity in biological systems; PNAS 98(24): 13763-13768). In summary, a specific protein sequence is easily determined from a known nucleic acid sequence, because the known sequence of nucleotides (i.e. a nucleic acid sequence) produces one and only one corresponding protein sequence (i.e. a sequence of amino acids); but, the inverse is not true because a specific sequence of nucleotides cannot be ascertained from a known sequence of amino acids since more than one distinct codon (i.e. set of three nucleotides) may code for a particular amino acid. Thus, a description of the amino acid per se, does not provide sufficient written description of the codon encoding that amino acid because the one-to-one correspondence is unidirectional, i.e., from codon forward to amino acid, but not from amino acid back-translated to codon. Thus, the specification does not adequately disclose the structures of the numerous nucleic acid sequences claimed by their capacity to encode the claimed protein sequence variants. Therefore, because the claimed functions cannot be predicted from the claimed partial structures or variations thereof, the functional characteristics must be determined empirically. Consequently, the claims are not enabled because even the skilled artisan cannot make and use the invention, with a reasonable expectation of success, without an undue amount of experimentation, based on the astronomically vast number of sequence variations permitted.
Relative Skill of Those in the Art: The relative level of skill of those in the art is deemed to be high (e.g. PhD level); however, even one of skill in the art could not predictably extrapolate the teachings in the specification, limited to the example in E. coli, to all of the sequence variants subsequently back translated into polynucleotides and then incorporated therein, with the same functional properties, as broadly as is claimed. The skilled artisan simply cannot envision the structures required, thus conception is not achieved until reduction to practice has occurred, regardless of the complexity or simplicity of the method used to determine such structures or to test for such properties, after the fact. Thus, even one of skill in the art, would have to engage in undue experimentation to determine which variations retain the necessary functional properties and thereby carry out the invention as claimed.
Quantity of Experimentation Necessary Based on Content of the Disclosure: The specification does not enable the genus because where the results are unpredictable, the disclosure of a single species (i.e. a single, well-defined sequence for a single protein to be back translated and incorporated into E. coli host cells) usually does not provide an adequate basis to support generic claims. This is because it is not apparent from the disclosure of one particular species, what other species will work; see MPEP 2164.03. One of skill in the art would neither expect nor predict the appropriate functioning of the numerous sequence variants back translated and incorporated, and accordingly, without such guidance, the experimentation left to those skilled in the art is unnecessarily and improperly extensive and undue. It is noted that providing methods for determining the functional properties, would not reduce the amount of experimentation required because the functional properties still must be determined empirically.
Therefore, in view of the lack of guidance and direction provided by Applicant there would be undue experimentation required to practice the claimed partial structures (e.g. amino acid sequence variations back translated into nucleic acids and incorporated into host cells), with a reasonable expectation of success, absent a specific and detailed description in Applicant's specification of how to effectively make and/or use the claimed invention. Thus, Applicant has not satisfied the requirements as set forth under 35 U.S.C. 112(a).
Applicant’s Arguments and Response to Arguments
11. All of Applicant’s arguments have been considered but were not deemed persuasive; accordingly, the rejection is maintained for reasons of record.
It is noted that Applicant put forth essentially the same arguments as addressed above (see Remarks, pages 11-14). However, similar to above, none of these arguments (including the declaration) are persuasive because they do not address the amount of experimentation required to make and use the invention, as now claimed.
Therefore, all of Applicant’s arguments have been considered (see responses above) but were not deemed persuasive; accordingly, the rejection is maintained for reasons of record and because when all of the evidence is considered, the totality of the rebuttal evidence of enablement fails to outweigh the evidence of lack of enablement (i.e. undue experimentation), especially with regards to newly added limitations encompassing the use of an unimaginable number of partial structures (e.g. as little as 70% sequence variations).
Conclusion
12. No claims are allowed.
13. THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
14. 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.
15. Any inquiry concerning this communication or earlier communications from the examiner should be directed to MARY MAILLE LYONS whose telephone number is (571)272-2966. The examiner can normally be reached on Monday-Friday 8 am to 5 pm EST. Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http: //www.uspto.gov/interviewpractice. If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Dan Kolker can be reached on (571)-272-3181. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/MARY MAILLE LYONS/Examiner, Art Unit 1645
March 26, 2026