DETAILED ACTION
Notice of Pre-AIA or AIA Status.0321
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Status
Claims 53, 59-66 & 69 are under examination on the merits.
Claims 67 & 68 are withdrawn from consideration as nonelected, without traverse, in response filed 02-06-2025.
Claims 1-52, 54-58 & 70 are cancelled.
Priority
Claims 53, 59-66 & 69 receive the foreign effective filing date of 6 February 2017.
Examiner’s Note
Prosecution is being reopened to address limitation of C-terminus residues remaining from cleavage of mitochondrial targeting protein (MTP).
Previous 112(b) rejection of claims 53, 59, 61 & 63 is withdrawn in view of Applicant’s claim amendments.
Previous 112(d) rejection of claims 53, 59, 61 & 63 is withdrawn in view of Applicant’s claim amendments.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 53, 59-66 & 69 are rejected under 35 U.S.C. 103 as being unpatentable over Donovan [PG Pub US 2016/0304842; Published 10-20-2016] in view of Smanski [Smanski et al. Nature Biotechnology. Volume 32, Number 12, December 2014; Published 11-24-2014] and in view of Niidome [J. Biol. Chem. 269: 24719–24722; Published 7 October 1994].
Due to Applicant’s amendment of the claims, the rejection is modified from that set forth in the Office action mailed 5 Feb 2026 as applied to claims 53, 59-66 & 69-70. Applicant’s arguments filed 4 June 2026 have been fully considered but are not persuasive.
Donovan teaches fusion proteins comprising Nif polypeptides in clusters, including combinations of the claimed NifK, NifD, NifH [p.1, ¶.8 & p.7, ¶.96]. They indicate combinations of Nif subunits and other elements can be made into fusion clusters including varied structural components that alter protein activity [¶.99].
They teach synthesis of Nif gene clusters is used to transfer nitrogenase activity to mitochondria via use of mitochondrial targeting proteins (MTPs) for expression of dinitrogenase reductase or dinitrogenase (i.e. making transgenic plants) [¶.80, 0105]. They teach which Nif enzymes are dinitrogenase reductase or dinitrogenase and list relevant associated enzymes [p.9, ¶.112; p.10, Tables 1 & 2].
They teach MTPs used in making such constructs [p.14, Tables 4 & 5]. They teach various length spacer sequences of nucleotides or amino acids can be used with MTP components to regulate activity, and that additions in the range of +2-8 additional residues from the end of the MTP can modify enzyme activity [0142]. Donovan teaches that a fusion protein with +8 residues between the targeting peptide and a nifH NP displays strong nitrogenase activity [p.14, Table 5].
Donovan teaches fusion proteins are cleaved (i.e. processed) from MTPs upon transport into mitochondria, releasing active subunits, such as NifD [¶.174, Fig.10].
They teach plants in which such constructs can be expressed, including wheat [p.7, ¶.101]. They teach via demonstration the expression of said constructs in tobacco plants [p.20, Example 12; p.11, ¶.123].
Donovan does not teach methods of generating fusion proteins involving NifZ. Donovan does not teach cleavage within the MTP wherein the C-terminal portions are directly fused to processed Nif subunits.
Smanski teaches iterative combination including NifZ components to engineer transgenic nitrogenase activity. In addition to the Nif subunits described by Donovan, Smanski includes NifZ and other combinatorial variations of the broader nitrogenase complex [p.1242, col.2, ¶.2]. Further, Smanski teaches how hundreds of such constructs can routinely be created and tested, directly stating that functionality assays can be tested with many different constructs [p.1245, col.1, ¶.3 & p.1248, col.1, ¶.4]. Smanski teaches that the genetic engineering of the nitrogenase pathway can be approached by iterative testing/optimization of various known structural components.
Smanski teaches that similar nitrogenase chimeric constructs have differential activity based solely on size of binding sites and/or spacer (i.e. sequence between subunit) length [p.1243, ¶.2]. Smanski teaches that one should make numerous variants of potential nitrogenase fusion constructs and vary structural elements as well as Nif and MTP subcomponents, including NifZ, while testing optimized expression within their target system [p.1248, ¶.1].
Niidome teaches that mitochondrial proteins are synthesized as larger precursors having N-terminal extension sequences required for targeting and localization to the mitochondria (i.e. comprise mitochondrial targeting peptides, MTPs) [p.2479, col.1, ¶.1]. They teach that following import, these extension sequences (i.e. MTPs) are proteolytically removed (i.e. processed or cleaved) in the mitochondrial matrix by mitochondrial processing peptidase (MPP) [id].
Niidome teaches that cleavage (i.e. processing) of N-terminal extension sequences (i.e. MTPs) by MPP occurs within the extension sequence itself, resulting in a series of residual amino acids remaining from the C-terminus end of the extension sequence (i.e MTP), attached to the N-terminus end of the fusion protein subunit intended to be transported into the mitochondrial matrix [p.24720, col.1, ¶.2, l.3-7; col.1, ¶.3—col.2, ¶.1; Figure 1]. They teach the processed fusion protein comprises residues remaining of the C-terminus end of the extension sequence (i.e. MTP) [Fig. 1].
Niidome additionally teaches that correct processing of proteins in mitochondria requires these extension peptides involved in cleavage and/or processing of proteins into active subunits [p.24719, col.1, ¶.1]. In their abstract they directly state the structure of these extensions as, “The minimal length of peptides for effective cleavage was…consisting of 11 and 5 residues from the cleavage site” (i.e. functional range of +5-11) [p.24719, Abstract].
Before the effective filing date of the claimed invention, it would have been obvious to one of skill in the art to modify methods of transgenic nitrogenases taught by Donovan utilizing routine optimization inclusive of NifZ, as described in Smanski. It would have been obvious to include and test various cleavage sites or MTP lengths necessary to the mitochondrial processing of proteins, because this is taught by Niidome as being critical to proper processing. This would include structures of Niidome’s ‘extension peptides’ (i.e. MTPs) that target synthetic fusion proteins to the mitochondria and are then cleaved/processed by MPP, leaving residual C-terminal portions of extension peptides.
One of ordinary skill in the art would have been motivated to combine these teachings because the ability to generate functional nitrogenase components in plant cells targeted to organelles, as described by Donovan for NifH, would allow one to overcome the known oxygen sensitivity of other Nif components of the nitrogenase system. It would be obvious to try various combinations of known Nif proteins, or potential subunit components, relevant to such a fusion polypeptide. It would be obvious to include cleavage/processing sites required for transport and processing of mitochondrial proteins, as described in Niidome.
This would include NifD, NifH, NifK and any supplemental Nif subcomponents that may enable or enhance nitrogenase activity, as outlined by Donovan. It would include the promoters or structural features required of a construct intended to be expressed in a plant. Smanski’s report would provide further specific teachings on how to optimize such combinations and additional nitrogenase components to be considered, such as NifZ. One would be motivated to combine these to achieve functional nitrogenase activity, involving additionally relevant Nif subunits, in plants.
Regarding claim 53 and amended claim 69, drawn to specific cleavage fragment sizing reciting a range of 5-11 amino acids; Niidome teaches the cleavage of ‘extension peptides’, corresponding to Applicant’s MTPs, that direct fusion proteins to the mitochondria for processing. Niidome teaches that MPP cleaves these extension peptides (i.e. MTPs), resulting in a residual C-terminus tail from the extension peptide to the processed product. Further, they teach that these residual fragments range from 5 to 11 residues in length [p.24720, col.1, ¶.2, l.3-7; col.1, ¶.3—col.2, ¶.1; Figure 1; p.24719, Abstract].
Moreover, all components limited to in the claims of the instant application (NifD, NifH, NifK, NifZ, MTPs, structural elements) are collectively described by Donovan and/or Smanski in creation of transgenic nitrogenase constructs. Smanski clearly teaches such constructs integrating NifZ, and that fusions of the nitrogenase complex would require routine optimization based on specific structural element changes, while providing the relevant methods of doing so. Donovan teaches the functional application of this in plants, whereby protection of the Nif complex subunits from oxidation is achieved by transport to the mitochondria, wherein such fusion proteins may be cleaved (i.e. processed) into functional subunits. Niidome describes MTPs, their cleavage characteristics, and C-terminus residuals from MTPs following processing.
One of ordinary skill in the art would have been motivated to perform such routine optimization work using the previously disclosed components because the primary hurdle to transferring engineered nitrogen-fixing ability to plants, broadly, is the protection of the Nif complex from oxidation in the cytosol and the optimization/increase of the activity of the enzymes involved. The prior art demonstrates methods of doing that via targeting to the mitochondria and combinatorial optimization of activity. Prior art also lays out the relevant components, methods, and structural considerations for iterative testing of such nitrogenase complex fusion proteins.
There would be strong motivation to combine these because the value of such transgenic nitrogen fixation in crop plants would be groundbreaking to agriculture and address a multitude of economic and environmental concerns surrounding the use of synthetic nitrogen fertilizers, as indicated by both Donovan [¶.6] and Smanski [p.1241, col.2, ¶.2].
Because of this, claims 53, 59-66 & 69 are obvious and are rejected.
Response to Arguments
Applicant urges (I) that description of processed products with +5-11 residues from the C-terminus of the MTP renders their invention non-obvious [Remarks, p.7, ¶.5]. Applicant remarks their invention is non-obvious because it comprises a cleavage fragment (CF) which is the Nif protein plus the additional +5-11 amino acid residues remaining of the C-terminus of the MTP following processing. Applicant argues Donovan and Smanski do not teach processed peptides having retained MTP C-terminus residues, thus making the claimed invention non-obvious [id].
This is unconvincing, as even Applicant admits Donovan teaches fusion proteins having MTP features that are cleaved (i.e. processed) following transport into the mitochondria [Remarks, p.8, .4]. This is graphically depicted in Donovan’s Figure 10, which shows cleaved fusion proteins [Donovan, ¶.27 & Fig.10]. Thus, Applicant points to Donovan’s teaching processed Nif polypeptides, present in the mitochondria of plants.
Further, the prior art of Niidome specifically describes processing of a fusion protein within the mitochondrial matrix, wherein cleavage of the MTP sequence occurs via MPP that results in a remnant C-terminal residues [see above, p.5, ¶.2—p.6, ¶.2].
Applicant urges (II) Donovan does not teach one to select a specific MTP construct from those they report [p.9, ¶.5]. Applicant points to Donovan’s teaching of 19 variant constructs using NifH, of which nine showed activity including those with +1, +2, +4, & +8 residues attached to NifH. Applicant admits the NifH+8 residue construct shows high activity [Remarks, p.10, .4].
Applicant’s argument is not persuasive because Donovan teaches such residue-possessing fusion proteins, having a Nif polypeptide +1-8 residues present after processing in the mitochondria, are active. Donovan uses NifH, one of the several potential Nif subunits, to demonstrate this. Donovan teaches activity of cleaved (i.e. processed) Nif subunits.
Additionally, in view of Niidome, it was known at the time of filing that cleavage of a fusion protein directly within the transport peptide sequence after import to the mitochondria would produce processed proteins having 5-11 amino acid residues remaining from the C-terminus of the MTP.
Donovan teaches that there is no clear trend in the number of residues and activity (i.e. it is unpredictable). One skilled in the art reading their statement would reasonably understand its context – that Donovan is teaching one needs to routinely test or validate residues of various lengths when optimizing activity of Nif fusions in plant organelles. This directs the reader to try various configurations, some of which, but not all, predictably work.
It is routine in molecular biology to test multiple sequences or construct forms for activity, particularly when the number of molecular variants to be tested relatively small. Because Donovan teaches there is no linear trend regarding the length: activity ratio, this informs the reader that it would also be prudent to test a range, at least including the range of values they show to work, and the residual C-terminus portions of MTPs taught by Niidome, when designing residue lengths.
Applicant urges (III) that Donovan uses the word ‘unpredictable’ and contends that this means one would not be able to predict that a Nif protein having +1-8 residues would show activity, or work [p.12, ¶.2].
However, this is not persuasive. The context of Donovan’s statement is that there is no clear, predictable trend in the relationship between number of residues and activity even though structural variants with +8 residues, and others, have activity. Donovan is indicating categories that potentially work.
That is, one cannot assume fewer number of residues (i.e. +1) provide less activity than a greater number (i.e. +8), and that there is no clear linear relationship between number of residues and activity. The teaching is that one cannot strictly rule out sequence lengths above or below a given threshold per se. They confirm that residues in the range of +1-8 provide activity, indicating that replicating their success using other Nif polypeptides would likely include routinely testing variants having at least +1-8 residues, or similar variants within a few residues of the high and low values.
Donovan does not need to explicitly state ‘other researchers should use variants with +8 residues’ verbatim because their data teaches this works, and one skilled in the art would recognize this. Their data clearly shows activity of Nif fusions having +1-8 residues attached. A skilled researcher would readily cross-apply this teaching of relevant residue or linker lengths used in Nif fusions.
Additionally, Niidome teaches the internal cleavage (i.e. processing) of the MTP portion of fusion proteins, which releases an active fusion having 5-11 residues of the C-terminus of the MTP attached to the processed fusion protein. Applicant’s claimed residue length is within this range.
This is exactly what Applicant does, albeit with the NifZ subunit and fusion variants having the indicated +1-8 residues, among others. Doing such is obvious cross-application of Donovan’s preliminary work, also informed by Niidome’s descripton of residual C-terminus sequences from MTPs, albeit iteratively testing alternative known Nif subunit(s) and residue lengths for effective mitochondrial targeting in plants.
Applicant yet further argues that the secondary Smanski reference is irrelevant to the instant Application, because Smanski only describes methods of engineering multi-unit Nif polypeptides in model hosts lacking mitochondria [Remarks, p.13, ¶.3]. Applicant also argues reference to Smanski is improper hindsight because Donovan does not directly refer to Smanksi [id].
However, the Smanski reference is included because it teaches that at the time of filing it was recognized that combinatorial testing of variant subunits, regulatory, or structural elements of Nif complexes was recognized as an effective optimization procedure (i.e. molecular fusion options, or chimeras). Smanski teaches NifZ, which is clearly relevant to the instant application. Further, if Donovan taught the exact constructs and residues as Applicant, the instant application would be anticipated rather than obvious. The current 103 rejection is made because Donovan lays out a clear path to the claimed invention, of which routine combinatorial testing of Nif fusions, as taught by Smanski as a secondary reference, leads to the variants included in claims of instant application.
A researcher concerned with transgenic engineering and optimization of Nif complexes in plants would reasonably be aware of the Smanski publication detailing such combinatorial approaches to optimizing nitrogenase activity in transgenic systems. The publication is not only directed to engineering of exogenous metabolic pathways, it is specifically relevant to engineering nitrogen fixation in plants. It details combinations and variations of Nif subunits. This is the core subject material of Applicant’s research. It is reasonable that one skilled in the art would understand Smanski’s teaching of iterative combination of features known to be important to nitrogen fixatoin, namely that one would want to test the at least +1-8 residues in conjunction with MTP described by Donovan.
Furthermore, the additional reference of Niidome points to the necessity and inclusion of such residual sequences adjacent to cleavage/processing sites in fusion proteins processed within the mitochondria. They directly point to a range of residue lengths of +5-11 as being the minimal standard for effective cleavage by mitochondrial processing protein and activity of mitochondrial peptides [p.24719, Abstract]. They describe such processed proteins as having residues of the C-terminus of the MTP which remain attached to the processed polypeptide [p.24720, col.1, ¶.2, l.3-7; col.1, ¶.3—col.2, ¶.1; Figure 1].
For these reasons, the previous obviousness rejection is maintained.
Conclusion
No claims are allowed.
Contact Information
Any inquiry concerning this communication or earlier communications from the examiner should be directed to KEITH R WILLIAMS whose telephone number is (571)272-3911. The examiner can normally be reached Mon - Fri, 9:30 - 5:30 EST.
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/KEITH R. WILLIAMS/Examiner, Art Unit 1663
/Amjad Abraham/SPE, Art Unit 1663
