DETAILED ACTION
Status of the Application
Claims 1-39 are pending.
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Applicant’s election of Group 26, claims 2-37, drawn in part to an engineered bacterium comprising genetic elements supporting programmable transcriptional activation and/or repression, wherein said genetic element comprises a nucleic acid encoding a fusion protein that comprises an RNA-binding protein and a transcriptional activator which is a SoxS family activator, wherein the RNA-binding protein is MCP, the election of a dCas9, the election of a J3 promoter, the election of the inducer m-toluic acid , and the election of p-aminocinnamic acid (p-ACA), as submitted in a communication filed on 5/12/2026 is acknowledged. Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.03(a)).
Claims 7-8, 32-36 are directed to non-elected inventions in view of the election of Group 26 and p-ACA. Claims 7-8, 32-36, 38-39 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 5/12/2026.
Claims 1 (linking claim), 2-6, 9-31, 37 are at issue and will be examined to the extent they encompass the elected invention.
Specification
The title of the invention is not descriptive. A new title is required that is clearly indicative of the invention to which the claims are directed. The claims under consideration are directed to a bacterium. Appropriate correction is required.
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code. See page 40, lines 13 and 18. Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code. See MPEP § 608.01.
Priority
Acknowledgment is made of a claim for domestic priority under 35 U.S.C. 119(e) to provisional application No. 63/335,143 filed on 04/26/2022.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 8/25/2023 is acknowledged. The submission is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Drawings
The drawings submitted on 4/25/2023 have been reviewed and are accepted by the Examiner for examination purposes.
Claim Objections
Claim 5 is objected to due to the recitation of “MCP”. Abbreviations unless otherwise obvious and/or commonly used in the art, should not be recited in the claims without at least once reciting the entire phrase for which the abbreviation is used. Appropriate correction is required.
Claim 27 is objected to due to the recitation of “encodes gene product”. To enhance clarity, the term should be amended to recite “encodes a gene product”. Appropriate correction is required.
Claim 28 is objected to due to the recitation of “PAL”. Abbreviations unless otherwise obvious and/or commonly used in the art, should not be recited in the claims without at least once reciting the entire phrase for which the abbreviation is used. If “PAL” refers to a “phenylalanine ammonia lyase”, this term should be recited at least once. Appropriate correction is required.
Claim Rejections - 35 USC § 112(b) or Second Paragraph (pre-AIA )
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 1-6, 9-31, 37 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 pre-AIA the applicant regards as the invention.
Claims 1 and 37 (claims 2-6, 9-31 dependent thereon) are indefinite in the recitation of “genetic elements supporting programable transcriptional activation and/or repression” for the following reasons. As written, it is unclear if the genetic elements required encompass genetic elements that encode transcriptional activators and/or repressors, or if the genetic elements are only required to encode elements that may assist in transcription activation and/or repression which are not necessarily transcriptional activators and/or repressors. Correction is required.
Claims 3 and 16 are indefinite in the recitation of “and the like” for the following reasons. This renders the claims indefinite because the claims include elements not actually disclosed (those encompassed by "and the like"), thereby rendering the scope of the claims unascertainable. See MPEP § 2173.05(d). For examination purposes, claim 3 will be interpreted as requiring any endonuclease lacking endonuclease activity, and claim 16 will be interpreted as requiring any vector. Correction is required.
Claim 5 is indefinite in the recitation of “SoxS-family activators” for the following reasons. It is unclear as to which activators are encompassed by the term. The specification states that an example of SoxS-family activators is the AraC/XylS family of activators. As known in the art, and also evidenced by Gallegos et al. (Microbiology and Molecular Biology Reviews 61(4):393-410, 1997), SoxS and TetD are part of this family. Claim 5 recites SoxS and TetD as a different effector domain from SoxS-family activators. Therefore, while the art/specification discloses SoxS and TetD as part of the SoxS-family activators by virtue of being part of the AraC/XylS family of activators, the claim appears to exclude these activators from the SoxS-family activators. For examination purposes, no patentable weight will be given to the term. Correction is required.
Claim 9 (claims 10-31 dependent thereon) is indefinite in the recitation of “wherein the at least one heterologous nucleic acid construct comprises a third nucleic acid sequence encoding a scaffold RNA” for the following reasons. Claim 9 depends from claim 2. Claim 2 refers to a first nucleic acid sequence in the nucleic acid construct. There is no mention of a second nucleic acid sequence in claim 2. Therefore, it is unclear as to how claim 9 could refer to a third nucleic acid sequence when there is no mention of a second nucleic acid sequence. In addition, it is unclear as to what is encompassed by the term “scaffold RNA”. The term does not convey a particular structure or some difference with any other type of RNA. For examination purposes, it will be assumed that the term simply refers to any RNA. Correction is required.
Claim 10 is indefinite in the recitation of “scRNA comprises a 3’ MS2 hairpin loop…” for the following reasons. It is unclear as to what a 3’ MS2 hairpin loop is. Does the term “3’” refers to a particular structural feature required in the MS2 hairpin loop? Does the term “3’” refer to the location of the MS2 hairpin loop in the scRNA, namely a MS2 hairpin loop located at the 3’ end of the scRNA? For examination purposes, it will be assumed that the RNA requires an MS2 hairpin loop. Correction is required.
Claim 11 (claims 12-31 dependent thereon) is indefinite in the recitation of “scRNA comprises a 5’ domain comprising a guide sequence that hybridizes to a target sequence” for the following reasons. It is unclear as to what a 5’ domain is. Does the term refer to an independent unknown domain which has been included in the scRNA? Does the term refer to a guide sequence which is located at the 5’ end of the scRNA? In addition, while a nucleic acid molecule can hybridize to another nucleic acid molecule, nucleotide sequences cannot hybridize to other nucleotide sequences in view of the fact that a nucleotide sequence is a graphical representation of the order in which nucleotides are arranged in a nucleic acid molecule. For examination purposes, it will be assumed that the RNA comprises a guide RNA that hybridizes to a target polynucleotide. Correction is required.
Claim 13 (claims 14-31 dependent thereon) is indefinite in the recitation of “wherein the at least one heterologous nucleic acid construct comprises a fourth nucleic acid sequence comprising an open reading frame of a gene of interest operatively linked to a promoter sequence and/or a PAM sequence, and wherein the target sequence is proximal…” for the following reasons. Claim 13 depends from claim 11, which depends from claim 9, which ultimately depends from claim 2. There is no mention of a second nucleic acid sequence in claim 9. Therefore, it is unclear as to how the construct of claim 13 can comprise a fourth nucleic acid sequence. For examination purposes, it will be assumed that the construct requires the nucleic acid encoding the endonuclease lacking endonuclease activity, the nucleic acid encoding an RNA that comprises a guide RNA that hybridizes to a target polynucleotide, and a nucleic acid encoding a protein of interest. Correction is required.
Claim 14 is indefinite in the recitation of “wherein the at least one heterologous nucleic acid construct comprises the first, second, third, and fourth sequences distributed in any combination on two vectors” for the following reasons. Claim 14 ultimately depends from claim 1. There is no mention in claim 2, claim 9 or claim 13 of a second sequence. Therefore, it is unclear as to which is the second sequence being referred to. In addition, the term “one heterologous nucleic acid construct” implies a single construct. If the sequences of the construct are spread between two plasmids, then there is no single construct. Correction is required.
Claim 15 is indefinite in the recitation of “the first, second, third and fourth sequences distributed on a single vector” for the following reasons. Claim 15 ultimately depends from claim 2. There is no mention in claim 2, claim 9 or claim 13 of a second sequence. Therefore, it is unclear as to which is the second sequence being referred to. For examination purposes, it will be assumed that claim 15 is directed to the engineered bacterium of claim 13 as interpreted above, wherein the construct is in a vector. Correction is required.
Claim 18 is indefinite in the recitation of “the first, second, third, and fourth sequences each comprise…” for the following reasons. . Claim 18 ultimately depends from claim 2. There is no mention in claim 2, claim 9 or claim 13 of a second sequence. Therefore, it is unclear as to which is the second sequence being referred to. For examination purposes, it will be assumed that claim 18 is directed to the engineered bacterium of claim 13 as interpreted above, wherein the construct is operably linked to a promoter. Correction is required.
Claim 20 (claims 23-26 dependent thereon) is indefinite in the recitation of “between about 60 to about 120 bases upstream …of a transcriptional start site …of the endogenous gene or open reading frame” for the following reasons. The term “about” encompasses a range which includes values which are higher and lower than the recited reference value (i.e., 60, 120). The term “between X to Y”, implies a range where X and Y are the endpoints. Therefore, in the absence of a clear definition of what is encompassed by the term “about”, the term "between about X to about Y" is unclear and confusing because X and Y are endpoints which are undefined ranges. In addition, there is no antecedent basis for the endogenous gene or open reading frame. For examination purposes, no patentable weight will be given to the term “and wherein the target sequence is between….or open reading frame”. Correction is required.
Claim 21 (claim 22 dependent thereon) is indefinite in the recitation of “wherein the target sequence is about 15 to about 25 bases upstream…of a transcriptional start site ..of the endogenous gene or open reading frame” for the following reasons. The term “about” encompasses a range which includes values which are higher and lower than the recited reference value (i.e., 15, 25). The term “is X to Y”, implies a range where X and Y are the endpoints. Therefore, in the absence of a clear definition of what is encompassed by the term “about”, the term " about X to about Y" is unclear and confusing because X and Y are endpoints which are undefined ranges. In addition, there is no antecedent basis for the endogenous gene or open reading frame. For examination purposes, it will be assumed that claim 21 is a duplicate of claim 13 as interpreted above. Correction is required.
Claim 22 is indefinite in the recitation of “wherein the target sequence corresponds with the …J3..promoter or portions thereof” for the following reasons. It is unclear as to how a target sequence, which is a nucleotide sequence can correspond to a promoter, which is a region of a polynucleotide where proteins bind to initiate transcription. As previously indicated, a nucleotide sequence is a graphical representation of the order in which nucleotides are arranged in a nucleic acid molecule. Therefore, it is unclear as to how a graphical representation can be equivalent to a promoter. In addition, the terms “J1”, “J3”, “J5” and “J6” are not well known promoters which convey a particular structure so that one of skill in the art would know what the structure of these promoters is or which promoters are encompassed or excluded from these terms. For examination purposes, it will be assumed that claim 22 is a duplicate of claim 13 as interpreted above. Correction is required.
Claim 23 is indefinite in the recitation of “the endogenous genes or open reading frame” for the following reasons. Claim 23 ultimately depends from claim 13. There is no antecedent basis for the endogenous genes or open reading frame. For examination purposes, it will be assumed that claim 23 is a duplicate of claim 20 as interpreted above. Correction is required.
Claim 24 is indefinite in the recitation of “…wherein the promoter sequence is a synthetic 5’-upstream sequence containing appropriate NGG PAM at an optimal position, wherein the optimal position is selected from about 75 to 85 nucleotides, about 78 to 83 nucleotides, and about 81 nucleotides upstream of the TSS” for the following reasons. It is unclear as to what a synthetic 5’-upstream sequence is. Does the term “5’” further limits the synthetic sequence structurally? Does the term “5’” refers to the location of the synthetic sequence at the 5’ end of the promoter?. In addition, the term “appropriate” is a relative term and the claim fails to provide the required reference to determine whether a PAM is appropriate or not. One cannot determine which PAMs are appropriate or not, and what are they appropriate for. For examination purposes, it will be assumed that claim 24 is a duplicate of claim 20 as interpreted above. Correction is required.
Claim 25 is indefinite in the recitation of “and related compounds” for the following reasons. In the absence of a clear definition of what is the relationship/property/characteristic associated with the term “related”, it is unclear as to what is encompassed by the term “related compounds”. Is a related compound, one that has the same molecular weight? Is a related compound one that has the same types of atoms?. For examination purposes, no patentable weight will be given to the term. Correction is required.
Claims 30 and 31 are indefinite in the recitation of “PapABC” and “AroGL” for the following reasons. The terms as written, appear to be generic and not limited to a specific organism. While the gene nomenclature used may be appropriate for. E. coli or P. fluorescens genes and proteins encoded by said genes, the use of this nomenclature for proteins of identical function from other organisms may not be accurate. As known in the art, genes encoding proteins of identical function in two different organisms may use different designations. For example, the ARO4 gene of Candida albicans encodes a DAHP synthase whereas the E. coli counterpart is the aroF gene. See the abstract of Sousa et al. (Microbiology 148(Pt5):1291-1303, 2002). As such, the use of terminology which is applicable to some organisms and not to others is confusing since the claims use this nomenclature with respect to any organism. As written, it is unclear if these terms intend to limit the proteins to proteins that are endogenously found in recombinant microorganism that use the gene nomenclature recited. If Applicant wishes to use the recited terminology in the claims, it is suggested that the claims be amended to clearly indicate the organism associated with the specific gene designation (e.g., E. coli AroGL). For examination purposes, the term “PapABC” will be interpreted as “4-amino-4-deoxychorismate synthase, 4-amino-4-deoxychorismate mutase, and 4-amino-4-deoxyprephenate dehydrogenase” while the term “AroGL” will be interpreted as “shikimate kinase II isozyme and 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) synthase isozyme”. Correction is required.
Claim 37 is indefinite in the recitation of “compounds with aromatic metabolites or intermediates” for the following reasons. It is unclear as to which intermediates are being referred to (e.g., intermediates of what?). In addition, a metabolite is a compound. Therefore, it is unclear as to what a compound with aromatic metabolites is. For examination purposes, no patentable weight will be given to the term “compounds with aromatic metabolites or intermediates”. Correction is required.
When amending the claims, applicant is advised to carefully review all examined claims and make the necessary changes to ensure proper antecedent basis and dependency.
Claim Rejections - 35 USC § 112(a) or First Paragraph (pre-AIA )
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 1-6, 9-31, 37 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 pre-AIA the inventor(s), at the time the application was filed, had possession of the claimed invention.
As stated in MPEP 2111.01, during examination, the claims must be interpreted as broadly as their terms reasonably allow. Claims 1-6, 9-31, 37 are directed in part to a genetically engineered bacterium that comprises a genus of heterologous genetic elements having any structure to assist in transcription activation and/or repression of any gene, endogenous or heterologous, wherein said genetic elements can include polynucleotides encoding a genus of endonucleases having any structure that lack endonuclease activity, a genus of Cas9 proteins having any structure which lack endonuclease activity, a genus of fusion proteins having any structure wherein said fusion proteins comprise any transcriptional activator and any RNA-binding protein, a genus of RNAs having any structure which are able to interact with any endonuclease and hybridize to any target polynucleotide, wherein the genetic elements can be under the control of any promoter induced by any small molecule, and wherein said genetically engineered bacterium can be engineered by any means to produce p-aminophenylalanine and/or p-aminocinnamic acid (p-ACA). See Claim Rejections - 35 USC § 112(b) or Second Paragraph (pre-AIA ) for claim interpretation.
In University of California v. Eli Lilly & Co., 43 USPQ2d 1938, the Court of Appeals for the Federal Circuit has held that “A written description of an invention involving a chemical genus, like a description of a chemical species, ‘requires a precise definition, such as by structure, formula, [or] chemical name,’ of the claimed subject matter sufficient to distinguish it from other materials”. As indicated in MPEP § 2163, 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, reduction to drawings, 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 that Applicant was in possession of the claimed genus. In addition, MPEP § 2163 states that a representative number of species means that the species which are adequately described are representative of the entire genus. Thus, when there is substantial variation within the genus, one must describe a sufficient variety of species to reflect the variation within the genus.
There is no actual structural limitation with regard to the members of the genus of heterologous genetic elements having any structure to assist in transcription activation and/or repression of any gene, the genus of endonucleases having any structure that lack endonuclease activity encoded by the recited polynucleotides, the genus of Cas9 proteins having any structure which lack endonuclease activity encoded by the recited polynucleotides, the genus of fusion proteins having any structure wherein said fusion proteins comprise any transcriptional activator and any RNA-binding protein encoded by the recited polynucleotides, the genus of RNAs having any structure which are able to interact with any endonuclease and hybridize to any target polynucleotide encoded by the recited polynucleotides, the genus of promoters induced by any small molecule, and the genus of genes encoding any enzyme associated with the production of p-AF and/or p-ACA required by the claims.
While the specification in the instant application discloses the structure of a single endonuclease lacking endonuclease activity (S. pyogenes dCas9), a limited number of RNA binding proteins, a limited number of transcriptional activators, a limited number of promoters that can be activated by the transcriptional activators disclosed, a limited number of genes encoding a phenylalanine ammonia lyase, a single gene operon from P. fluorescens encoding enzymes associated with the production of p-aminocinnamic acid and/or p-aminophenylalanine, a single aroGL operon from E. coli, and a single promoter induced by a small molecule (XylS/Pm from P. putida mt-2), it provides no clue as to the structural elements required in any heterologous genetic element that can assist in transcription activation and/or repression of any gene, any endonuclease that lack endonuclease activity, any Cas9 protein which lack endonuclease activity, any fusion protein that comprises any transcriptional activator and any RNA-binding protein, any RNA which is able to interact with any endonuclease and hybridize to any target polynucleotide, any promoter induced by any small molecule, and any enzyme associated with the production of p-AF and/or p-ACA, including any 4-amino-4-deoxychorismate synthase, any 4-amino-4-deoxychorismate mutase, any 4-amino-4-deoxyprephenate dehydrogenase, any shikimate kinase II isozyme and any 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) synthase isozyme. No disclosure of a structure/function correlation has been provided which would allow one of skill in the art to recognize which polynucleotides have the desired activity or encode the desired protein/RNA.
The claims require a large genus of nucleic acids which are structurally unrelated. A sufficient written description of a genus of polynucleotides and polypeptides may be achieved by a recitation of a representative number of polynucleotides and polypeptides defined by their nucleotide and amino acid sequences, respectively, or a recitation of structural features common to members of the genus, which features constitute a substantial portion of the genus. However, in the instant case, there is no structural feature which is representative of all the members of the genus of nucleic acids recited in the claims, or the members of the genus of proteins/RNAs encoded by the recited genus of nucleic acids, and there is no information as to a correlation between structure and function. Furthermore, while one could argue that the few species disclosed are representative of the structure of all the members of the genus, it is noted that the art teaches several examples of how even highly structurally homologous polypeptides can have different enzymatic activities. For example, Witkowski et al. (Biochemistry 38:11643-11650, 1999) teach that one conservative amino acid substitution transforms a β-ketoacyl synthase into a malonyl decarboxylase and completely eliminates β-ketoacyl synthase activity. Tang et al. (Phil Trans R Soc B 368:20120318, 1-10, 2013) teach that two Dehalobacter reductive dehalogenases, CfrA and DcrA, having 95.2% sequence identity to teach other have exclusively different substrates (Abstract; page 7, left column, Discussion, CfrA and DcrA). Seffernick et al. (J. Bacteriol. 183(8):2405-2410, 2001) teach that two naturally occurring Pseudomonas enzymes having 98% amino acid sequence identity catalyze two different reactions: deamination and dehalogenation, therefore having different function. Therefore, since minor structural differences may result in changes affecting function, and no additional information correlating structure with the desired functional characteristics has been provided, one cannot reasonably conclude that the few species disclosed are representative of the structure of all the proteins encoded by the nucleic acids of the claims.
Due to the fact that the specification only discloses a limited number of species of the genus, and the lack of description of any additional species by any relevant, identifying characteristics or properties, one of skill in the art would not recognize from the disclosure that Applicant was in possession of the claimed invention.
Claim 16 rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the enablement requirement. The claim(s) 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.
Claim 16 appears to require novel vectors (e.g., pBBR1, pRK2, pRSF1010, pBAV1). Since the vectors are essential to the claimed invention, they must be obtainable by a repeatable method set forth in the specification or otherwise be readily available to the public. The recited vectors’ sequences are not fully disclosed, nor have all the sequences required for their construction been shown to be publicly known and freely available. The enablement requirements of 35 U.S.C. § 112 may be satisfied by a deposit of the vectors. The specification does not disclose a repeatable process to obtain the vectors and it is not apparent if the DNA sequences are readily available to the public. Accordingly, it is deemed that a deposit of these vectors should have been made in accordance with 37 CFR 1.801-1.809.
If a deposit was made under the terms of the Budapest Treaty, then an affidavit or declaration by applicants, or a statement by an attorney of record over his or her signature and registration number, stating that the specific strain has been deposited under the Budapest Treaty and that the strain will be available to the public under the conditions specified in 37 CFR 1.808, would satisfy the deposit requirement made herein.
If a deposit has not been made under the Budapest treaty, then in order to certify that the deposit meets the criteria set forth in 37 CFR 1.801-1.809, applicants may provide assurance or compliance by an affidavit or declaration, or by a statement by an attorney of record over his or her signature and registration number, showing that:
a. during the pendency of this application , access to the invention will be afforded to the Commissioner upon request;
b. upon granting of the patent the strain will be available to the public under the conditions specified in 37 CFR 1.808;
c. the deposit will be maintained in a public repository for a period of 30 years or 5 years after the last request or for the effective life of the patent, whichever is longer; and
d. the deposit will be replaced if it should ever become unviable.
Claims 1-6, 9-31, 37 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 (A) a recombinant engineered bacterium that has been transformed with (i) a nucleic acid encoding a R. glutinis phenylalanine ammonia lyase, (ii) a P. fluorescens papABC operon, and (iii) an E. coli aroGL operon, wherein said recombinant engineered bacterium further comprises (a) a nucleic acid encoding a S. pyogenes dCas9 protein, (b) a nucleic acid encoding a fusion protein comprising the RNA binding protein MCS and the transcription activator SoxS, and (c) a nucleic acid encoding an RNA that comprises a guide RNA that can hybridize to a promoter operably linked to the nucleic acid of (i) or the nucleic acid of (ii), wherein said RNA also comprise an MS2 hairpin loop that interacts with SoxS, and wherein the guide RNA forms a complex with the dCas9 protein, wherein the nucleic acid of (i), (ii), (a), (b) or (c) is operably linked to the XylS/Pm promoter from P. putida mt-2, and (B) a composition comprising the recombinant engineered bacterium of (A) and a growth medium, does not reasonably provide enablement for a genetically engineered bacterium that comprises heterologous genetic elements having any structure to assist in transcription activation and/or repression of any gene, endogenous or heterologous, wherein said genetic elements can include polynucleotides encoding endonucleases having any structure that lack endonuclease activity, Cas9 proteins having any structure which lack endonuclease activity, fusion proteins having any structure wherein said fusion proteins comprise any transcriptional activator and any RNA-binding protein, RNAs having any structure which are able to interact with any endonuclease and hybridize to any target polynucleotide, wherein the genetic elements can be under the control of any promoter induced by any small molecule, and wherein said genetically engineered bacterium can be engineered by any means to produce p-aminophenylalanine (p-AF) and/or p-aminocinnamic acid (p-ACA). 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 and/or use the invention commensurate in scope with these claims.
Factors to be considered in determining whether undue experimentation is required are summarized in In re Wands (858 F.2d 731, 737, 8 USPQ2nd 1400 (Fed. Cir. 1988)) as follows: 1) quantity of experimentation necessary, 2) the amount of direction or guidance presented, 3) the presence and absence of working examples, 4) the nature of the invention, 5) the state of prior art, 6) the relative skill of those in the art, 7) the predictability or unpredictability of the art, and 8) the breadth of the claims. The factors which have led the Examiner to conclude that the specification fails to teach how to make and/or use the claimed invention without undue experimentation, are addressed in detail below.
The breadth of the claims. Claims 1-6, 9-31, 37 broadly encompass a genetically engineered bacterium that comprises heterologous genetic elements having any structure to assist in transcription activation and/or repression of any gene, endogenous or heterologous, wherein said genetic elements can include polynucleotides encoding endonucleases having any structure that lack endonuclease activity, Cas9 proteins having any structure which lack endonuclease activity, fusion proteins having any structure wherein said fusion proteins comprise any transcriptional activator and any RNA-binding protein, RNAs having any structure which are able to interact with any endonuclease and hybridize to any target polynucleotide, wherein the genetic elements can be under the control of any promoter induced by any small molecule, and wherein said genetically engineered bacterium can be engineered by any means to produce p-aminophenylalanine (p-AF) and/or p-aminocinnamic acid (p-ACA).
The enablement provided is not commensurate in scope with the claims due to the extremely large number of nucleic acids of unknown structure encompassed by the claims. In the instant case, the specification enables (A) a recombinant engineered bacterium that has been transformed with (i) a nucleic acid encoding a R. glutinis phenylalanine ammonia lyase, (ii) a P. fluorescens papABC operon, and (iii) an E. coli aroGL operon, wherein said recombinant engineered bacterium further comprises (a) a nucleic acid encoding a S. pyogenes dCas9 protein, (b) a nucleic acid encoding a fusion protein comprising the RNA binding protein MCS and the transcription activator SoxS, and (c) a nucleic acid encoding an RNA that comprises a guide RNA that can hybridize to a promoter operably linked to the nucleic acid of (i) or the nucleic acid of (ii), wherein said RNA also comprise an MS2 hairpin loop that interacts with SoxS, and wherein the guide RNA forms a complex with the dCas9 protein, wherein the nucleic acid of (i), (ii), (a), (b) or (c) is operably linked to the XylS/Pm promoter from P. putida mt-2, and (B) a composition comprising the recombinant engineered bacterium of (A) and a growth medium.
The amount of direction or guidance presented and the existence of working examples. The specification discloses the structure of a single endonuclease lacking endonuclease activity (S. pyogenes dCas9), a limited number of RNA binding proteins, a limited number of transcriptional activators, a limited number of promoters that can be activated by the transcriptional activators disclosed, a limited number of genes encoding a phenylalanine ammonia lyase, a single gene operon from P. fluorescens encoding enzymes associated with the production of p-aminocinnamic acid and/or p-aminophenylalanine, a single aroGL operon from E. coli, and a single promoter induced by a small molecule (XylS/Pm from P. putida mt-2). However, the specification does not provide information as to the structural elements required in any heterologous genetic element that can assist in transcription activation and/or repression of any gene, any endonuclease that lack endonuclease activity, any Cas9 protein which lack endonuclease activity, any fusion protein that comprises any transcriptional activator and any RNA-binding protein, any RNA which is able to interact with any endonuclease and hybridize to any target polynucleotide, any promoter induced by any small molecule, and any enzyme associated with the production of p-AF and/or p-ACA, including any 4-amino-4-deoxychorismate synthase, any 4-amino-4-deoxychorismate mutase, any 4-amino-4-deoxyprephenate dehydrogenase, any shikimate kinase II isozyme and any 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) synthase isozyme. No disclosure of a structure/function correlation has been provided which would allow one of skill in the art to recognize which polynucleotides have the desired activity or encode the desired protein/RNA.
The state of prior art, the relative skill of those in the art, and the predictability or unpredictability of the art. The coding region of a polynucleotide determines the structure and function of the protein encoded by said polynucleotide. While the art discloses a limited number of polynucleotides that encode endonucleases having no enzymatic activity, a limited number of Cas9 proteins that lack endonuclease activity, a limited number of transcription activators, a limited number of RNA binding proteins, a limited number of 4-amino-4-deoxychorismate synthases, 4-amino-4-deoxychorismate mutases, 4-amino-4-deoxyprephenate dehydrogenases, shikimate kinase II isozymes and DAHP synthase isozymes, a limited number of promoters that are induced by small molecules, and a limited number of RNAs that can bind to an endonuclease, the art is silent with regard to the structural features required in the recited polynucleotides, or a structure/function correlation that would allow one of skill in the art to recognize those nucleic acids required by the claims.
The art clearly teaches that (a) determining function based solely on structural homology, and (b) modification of a protein’s amino acid sequence to obtain the desired activity without any guidance/knowledge as to which amino acids in a protein are tolerant of modification and which ones are conserved are highly unpredictable. For example, Singh et al. (Current Protein and Peptide Science 19(1):5-15, 2018) disclose different protein engineering approaches and state that despite the availability of an ever-growing database of protein structures and highly sophisticated computational algorithms, protein engineering is still limited by the incomplete understanding of protein functions, folding, flexibility and conformational changes (page 11, left column, last paragraph). Sadowski et al. (Current Opinion in Structural Biology 19:357-362, 2009) teach that much of the problem in assigning function from structure comes from functional convergence, where although a stable structure is required to perform many functions it is not always necessary to adopt a particular structure to carry out a particular function (page 357, right column, first full paragraph). Sadowski et al. further explain that the unexpected and significant difficulties of predicting function from structure show that the potential of structural models for providing novel functional annotations has not yet fully realized. Sadowski et al. also states that while a few successes have been achieved which required manual intervention, the ability to vary the requirements for specificity in prediction means that it is difficult to determine how useful the end result may be for the user (page 361, left column, first full paragraph). The teachings of Singh et al. and Sadowski et al. are further supported by the teachings of Witkowski et al., Tang et al. and Seffernick et al. already discussed above, where it is shown that even small amino acid changes result in enzymatic activity changes.
The quantity of experimentation required to practice the claimed invention based on the teachings of the specification. While methods of generating or isolating variants of a polynucleotide or a polypeptide as well as assays were known in the art at the time of the invention, it was not routine in the art to screen by a trial and error process for an essentially infinite number of polynucleotides to find those encoding the desired proteins/RNAs or those that are promoters or genetic elements that help in transcriptional activation and/or repression of any gene. In the absence of (i) a rational and predictable scheme for selecting those nucleic acids most likely to have the desired functional features, and/or (ii) a correlation between structure and the desired activity, one of skill in the art would have to test an essentially infinite number of proteins to determine which ones have the desired functional characteristics.
Therefore, taking into consideration the extremely broad scope of the claim, the lack of guidance, the amount of information provided, the lack of knowledge about a correlation between structure and the desired function, and the high degree of unpredictability of the prior art in regard to structural changes and their effect on function, one of ordinary skill in the art would have to go through the burden of undue experimentation in order to practice the claimed invention. Thus, Applicant has not provided sufficient guidance to enable one of ordinary skill in the art to make and use the invention in a manner reasonably correlated with the scope of the claims.
Claim Rejections - 35 USC § 102 (AIA )
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale or otherwise available to the public before the effective filing date of the claimed invention.
Claims 1-6, 9-18, 21-22, 27, 37 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Fontana et al. (Nature Communications 11(1618), pages 1-11, published online 4/1/2020).
Claims 1-6, 9-18, 21-22, 27, 37 are directed in part to (i) an engineered bacterium that can produce an aromatic compound, wherein said bacterium comprises a heterologous nucleic acid construct that comprises (a) a nucleic acid that encodes a dCas9 protein, (b) a transcriptional activator which is a fusion of the RNA binding protein MCP and the effector domain SoxS, and (c) an RNA that comprises a guide RNA that hybridizes to a target nucleic acid and a MS2 hairpin loop, wherein the target nucleic acid is proximal to a PAM and/or a promoter, wherein the heterologous nucleic acid construct can also further comprise a nucleic acid encoding a protein of interest operably linked to a promoter and/or a PAM, wherein the heterologous construct can be in a vector or integrated into the genome of the engineered bacterium, wherein the construct is operably linked to a promoter operable in the engineered bacterium, and wherein the engineered bacterium comprises a gene encoding a protein that results in the production of an aromatic compound, and (ii) a system for the production of aromatic compounds, wherein said system that comprises the engineered bacterium of (a) and a growth medium..
Fontana et al. teach an E. coli cell which comprises a heterologous nucleic acid construct that comprises a nucleic acid that encodes a dCas9, a nucleic acid encoding a fusion protein that comprises an RNA binding domain MCP and the transcription effector SoxS, and a nucleic acid encoding an RNA that comprises a guide RNA and a MS2 hairpin loop (scRNA; page 2, left column). Fontana et al. teach that the scRNA hybridizes to a promoter upstream of an endogenous aroK-aroB operon which encodes proteins associated with the production of aromatic amino acids (page 2, right column; page 3, Figure 2a). Fontana et al. also teach an E. coli cell which comprises a heterologous nucleic acid construct that comprises a nucleic acid that encodes a dCas9, a nucleic acid encoding a fusion protein that comprises an RNA binding domain MCP and the transcription effector SoxS, and a nucleic acid encoding an RNA that comprises a guide RNA and a MS2 hairpin loop, wherein the scRNA hybridizes to a J3 promoter that is operably linked to a heterologous gene (mRFP1 and sfGFP; page 5, Figure 3 caption; page 2, Figure 1a). Fontana et al. teach the integration of the mRFP1 and sfGFP genes (page 2, Figure 1; Supplemental information, page 17). Fontana et al. teach that the nucleic acids encoding the dCas9, the MCP-SoxS fusion and the scRNA are operably linked to promoters and vectors comprising said nucleic acids including those encoding the heterologous gene operably linked to promoters (page 9, Bacterial strain construction and manipulation). Since the E. coli cell endogenously produces aromatic amino acids, it follows that the engineered E. coli cell of Fontana et al. would produce aromatic compounds when cultured. Fontana et al. teach culturing the recombinant E. coli cell in a culture medium (page 9, right column), thus teaching a system that produces aromatic compounds which comprises an engineered bacterium that comprises genetic elements to support transcription activation and a growth medium. Therefore, the teachings of Fontana et al. anticipate the instant claims as written/interpreted.
Claims 1-6, 9-31, 37 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Faulkner (Ph.D. Dissertation, CRISPR-based Control of Biosynthetic Pathways and Genetic Biosensors, Informed by a Quantitative Investigation of Metabolic Burden, University of Washington, 2021).
Claims 1-6, 9-24, 27-31, 37 are directed in part to (i) an engineered bacterium, wherein the engineered bacterium can be P. putida, wherein said bacterium comprises a heterologous nucleic acid construct that comprises (a) a nucleic acid that encodes a dCas9 protein, (b) a transcriptional activator which is a fusion of the RNA binding protein MCP and the effector domain SoxS, (c) an RNA that comprises a guide RNA that hybridizes to a target nucleic acid and a MS2 hairpin loop, wherein the target nucleic acid is proximal to a PAM and/or a promoter, wherein the heterologous nucleic acid construct can also further comprise a nucleic acid encoding a protein of interest operably linked to a promoter and/or a PAM, wherein the heterologous construct can be in a vector or integrated into the genome of the engineered bacterium, wherein the construct is operably linked to a promoter operable in the engineered bacterium, wherein the protein of interest is a protein that results in the production of p-aminocinnamic acid (p-ACA), wherein the engineered bacterium can comprise a nucleic acid that encodes a phenylalanine ammonia lyase, a 4-amino-4-deoxychorismate synthase, a 4-amino-4-deoxychorismate mutase, a 4-amino-4-deoxyprephenate dehydrogenase, a shikimate kinase II isozyme and 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) synthase isozyme, and (b) a system for the production of aromatic compounds, wherein said system that comprises the engineered bacterium of (a) and a growth medium..
Faulkner teaches a recombinant P. putida that produces p-ACA that comprises a nucleic acid construct integrated in the genome of the recombinant P. putida wherein said construct comprises a nucleic acid encoding a dCas9, and a nucleic acid encoding a MCP-SoxS fusion, wherein said recombinant P. putida was further transformed with a plasmid that encodes a RNA that comprises a guide RNA that hybridizes to a promoter region and has an MS2 hairpin loop, and a plasmid that comprises a nucleic acid that encodes a phenylalanine ammonia lyase (PAL), a P. fluorescens papABC operon and an E. coli aroGL operon (pages 71, 72, 80, 85-87, page 88, Figure 3.S2). The construct comprises promoters to express the nucleic acids encoding the dCas9, the MCP-SoxP fusion, the RNA, and the PAL. P. putida endogenously produces amino acids, including aromatic amino acids, thus comprising a gene that encodes a protein that results in the production of an aromatic compound. Faulkner teaches culturing the recombinant P. putida in culture medium, thus teaching a system for the production of aromatic compounds that comprises an engineered bacterium and culture medium (pages 86-87). Therefore, the teachings of Faulkner anticipates the instant claims as written/interpreted.
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
Conclusion
No claim is in condition for allowance.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
Applicant is advised that any Internet email communication by the Examiner has to be authorized by Applicant in written form. See MPEP § 502.03 (II). Without a written authorization by Applicant in place, the USPTO will not respond via Internet email to any Internet correspondence which contains information subject to the confidentiality requirement as set forth in 35 U.S.C. 122. Sample written authorization language can be found in MPEP § 502.03 (II). An Authorization for Internet Communications in a Patent Application or Request to Withdraw Authorization for Internet Communications form (SB/439) can be found at https://www.uspto.gov/patent/forms/ forms-patent-applications-filed-or-after-september-16-2012, which can be electronically filed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to DELIA M RAMIREZ, Ph.D., whose telephone number is (571) 272-0938. The examiner can normally be reached on Monday-Friday from 8:30 AM to 5:00 PM. If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Robert B. Mondesi, can be reached at (408) 918-7584. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
/DELIA M RAMIREZ/Primary Examiner, Art Unit 1652
DR
August 2, 2026
Prosecution Insights