DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Status
Claims 1-20 are pending.
Claims 1-20 are examined on the merits.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1-3, 5-14, and 16-18 rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claims 1 and 12 recite “aspartic protease AED3-1”. Although the specification identifies and characterizes a particular AED3-1 protein, the metes and bounds of the term as recited in claim 1 and 12 are unclear.
Paragraph [0078] defines “AED3-1”, “aspartic protease AED3-1”, and “aspartic protease AED3-like” as “aspartyl protease apoplastic enhanced disease susceptibility 3-1”, also referred to as “aspartic protease 53” or “AP53”. Paragraphs [0052, 00178, 00192] and Table 2 identify the experimentally studied Nicotiana benthamiana protein as LOC107806163 and UniProt Accession No. A0A1S4BAC6, and paragraphs [0090-0093] disclose embodiments comprising SEQ ID NO: 1 or sequences having specified degrees of identity to SEQ ID NO:1.
However, paragraph [0078] further states that AED3-1 may be from “any plant or plant cell” and only optionally corresponds to UniProt Accession No. A0A1S4BAC6. The sequence and sequence-identity disclosures are presented as embodiments and are not incorporated into claims 1 and 12. Accordingly, claims 1 and 12 do not require LOC107806163 and UniProt Accession No. A0A1S4BAC6, SEQ ID NO: 1, any minimum degree of sequence identity, identity over the entire protein, an orthologous relationship, a conserved structural feature, or a defined biochemical or biological activity.
The specification further demonstrates that the designation cannot be determined merely from membership in the broader aspartic-protease family. Paragraphs [0052, 00178, 00192] separately identify AP A1, ADE3-1, and AED3-2 as distinct aspartic proteases. Moreover, silencing AED3-1 increased trastuzumab production, whereas silencing AED3-2 or AP A1 reduced trastuzumab production, and combined silencing of AED3-1 and AED3-2 did not significantly increase production. Thus, the specification establishes that different related aspartic proteases are not functionally interchangeable, but it does not identify the structural, phylogenetic, or functional criteria by which a skilled artisan would determine which proteins from any plant species qualify as AED3-1.
The state of the art likewise does not provide a sufficiently definite boundary. General classification as plant aspartic protease, Family A1 protease, atypical aspartic protic protease, or Clan AA member encompasses numerous distinct proteins and does not distinguish AED3-1 from AED3-2, AP A1, or another aspartic protease. Therefore, those general classifications do not resolve the ambiguity created by the unrestricted cross-species language in paragraph [0078].
Accordingly, one of ordinary skill in the art would not be able to determine with reasonable certainty whether “aspartic protease AED3-1” in claims 1 and 12 is limited to LOC107806163 or the protein represented by SEQ ID NO: 1 and UniProt Accession No. A0A1S4BAC6, encompasses orthologs satisfying an unstated structural of functional criterion, or includes additional undefined aspartic protease from any plant or plant cell.
Dependent claims 2-3, 5-11, 13, 14, and 16-18 are included in this rejection because they do not include additional limitations to resolve the ambiguity.
Claim Rejections - 35 USC § 112
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.
Scope of Enablement
Claims 4-7, 12, and 15-20 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specifications, while being enabling for inhibiting the specific AED3-1 gene identified by SEQ ID NO:1 and the specific inhibitory nucleotide identified by SEQ NO:2, including the particular embodiments described in the Examples, does not reasonably provide enablement for the full scope of the claimed invention, namely inhibition of AED3-1 by any inhibitor, including polypeptides, small molecule drugs, shRNAs, siRNAs, miRNAs, ASOs, functional variants of SEQ ID NO: 2, genomic modifications conferring reduced or loss of function, and, with respect to claim 4 and 15, AED3-1 amino acid sequence having at lease about 60% sequence identity to SEQ ID NO: 1. 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.
An “analysis of whether a particular claim is supported by the disclosure in an application requires a determination of whether that disclosure, when filed, contained sufficient information regarding the subject matter of the claims as to enable one skilled in the pertinent art to make and use the claimed invention.” MPEP 2164.01. “A conclusion of lack of enablement means that. . . the specification, at the time the application was filed, would not have taught one skilled in the art how to make and/or use the full scope of the claimed invention [i.e. commensurate scope] without undue experimentation.” In re Wright, 999 F.2d 1557,1562, 27 USPQ2d 1510, 1513 (Fed. Cir. 1993); MPEP 2164.01.
In In re Wands, 858 F.2d 731,8 USPQ2d 1400 (Fed. Cir. 1988), several factors implicated in determination of whether a disclosure satisfies the enablement requirement and whether any necessary experimentation is “undue” are identified. These factors include, but are not limited to:
(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, 858 F.2d 731,737, 8 USPQ2d 1400, 1404 (Fed. Cir. 1988). No single factor is independently determinative of enablement; rather “[i]t is improper to conclude that a disclosure is not enabling based on an analysis of only one of the above factors while ignoring one or more of the others.” MPEP 2164.01. Likewise, all factors may not be relevant to the enablement analysis of any individual claim.
Claims 4-7, 12, and 15-20 are not limited to the specific shRNA-mediated inhibition of AED3-1 disclosed in the specification. Rather, the claims encompass inhibition of AED3-1 using numerous distinct mechanisms, including polypeptides, small-molecule drugs, shRNAs, siRNAs, miRNAs, antisense oligonucleotides, functional variants of SEQ ID NO: 2, genomic modifications conferring reduced or loss of function, and , for claims 4 and 15, AED3-1 proteins having at least about 60% sequence identity to SEQ ID nO:1. The claims further encompass numerous heterologous proteins and plant species.
Under the broadest reasonable interpretation consistent with the specification, the claimed term “AED3-1” is not limited to the specific Nicotiana benthamiana AED3-1 protein (LOC107806163 and UniProt Accession No. A0A1S4BAC6, or SEQ ID NO: 1) exemplified in specification. Rather, the specification expressly uses the terms “AED3-1”, “aspartic protease AED3-1”, and “aspartic protease AED3-like”, indicating that the claimed term encompasses the corresponding AED3 (AED3-like) aspartic protease in plants, including orthologous or homologous AED3 proteins from other plant species, and is not restricted to the single exemplified sequence. Wang’s (Bing Wang et. al., International Journal of Biological Macromolecules (2024) 279, 135016, pp1-14) genome-wide analysis identifies numerous members of tobacco aspartic-protease family but does not identify the entire family, or each member thereof, as “AED3-1” (Fig 1, p4 “Result”). Thus, “AED3-1” is not shown to be an art-recognized synonym for any plant aspartic protease.
Although a person of ordinary skill in the art would have been familiar with plant molecular biology, recombinant protein expression, RNA-mediated gene silencing, and genome-editing techniques, the biological effects of inhibiting individual plant aspartic protease were not reasonably predictable. Wang explains, plant aspartic proteases comprise a large and functionally diverse protein family whose members differ in expression, localization, and biological function (Abstract, Fig 1). Thus, even within the broader AED3-like proteins encompassed by the claims, a person of ordinary skill in the art would not have reasonably predicted which members could inhibited to achieve the claimed increase in recombinant protein production without undue experimentation. The unpredictability is further demonstrated by the specification itself. Silencing AED3-1 increased trastuzumab production, whereas silencing the related AED3-1 or AP A1 decreased trastuzumab production, and simultaneous silencing of AED3-1 and AED3-2 did not significantly increase production. These differing outcomes demonstrate that the effect of inhibiting a particular plant aspartic protease on heterologous protein production could not have been reliably predicted. Accordingly, a person of ordinary skill in the art would not have reasonably predicted that inhibition of a particular AED3-1 protein, or proteins across the full scope of the claims using the broad range of claimed inhibitor classes or genomic modifications, would enhance heterologous protein production without substantial experimentation. The unpredictability of the art is further demonstrated by the specification itself. Silencing AED3-1 increased trastuzumab production, whereas silencing the related AED3-2 or AP A1 decreased trastuzumab production, and simultaneous silencing of AED3-1 and AED3-2 die not significantly increase production. These differing outcomes would not have permitted a person of ordinary skill in the art to extrapolate the single successful embodiment to the full scope of the claimed invention without undue experimentation.
In contrast, the specification describes enhancing heterologous protein production in Nicotiana benthamiana by reducing the activity of the endogenous aspartic protease AED3-1. The disclosure is centered on a single experimental approach in which a specific shRNA targeting AED3-1 is transiently expressed by Agrobacterium-mediated agroinfiltration together with a trastuzumab expression construct. The working examples evaluate trastuzumab accumulation approximately seven days after infiltration and demonstrate improved protein production following shRNA-mediated silencing of AED3-1. Aside from this specific embodiment, the specification provides no working examples demonstration other inhibitor classes, genomic loss-of-function modification, broad AED3-1 sequence variants, or functional variants of the disclosed inhibitory sequence.
The working example is limited to post-transcriptional gene silencing of AED3-1 using shRNA. The specification describes that shRNAs were designed complementary to selected aspartic protease, including AED3-1/LOC107806163, cloned into a plastid, transformed into Agrobacterium tumefaciens, and co-infiltrated with pPFC0058 into N. benthamiana plants. The plants were grown for 7 days post-infiltration, and trastuzumab production was measured. The specification reports that shRNA-mediated silencing of AED3-1 increased trastuzumab production.
However, claims 5-7, 12, and 16-17 are not limited to this demonstrated shRNA embodiment. Claim 5 broadly recited contacting the plant or plant cell with an inhibiter of AED3-1. Claims 6 and 16 further encompass inhibitors including polypeptides, small molecule drugs, shRNA, miRNA, ASO, and nucleotides comprising SEQ ID NO: 2 or functional variants thereof. The specification identities these inhibitor classes generally, but does not provide working examples, representative species, screening assays, structures, concentration’s, delivery conditions, or guidance sufficient to identify and use polypeptide inhibitors, small molecule inhibitors, siRNA inhibitors, miRNA inhibitors or ASO inhibitors that inhibit AED3-1 and enhance heterologous protein production across the full scope of the claims.
Claims 7, 12 and 17 also encompass genomic modifications in an AED3-1 gene that confer reduced or loss of function, including insertions, or substitutions. The specification generally states that such genomic modifications may be introduced by DRISPR/Cas, TALEN, ZFN, physical mutagenesis, or chemical mutagenesis. However, the specification does not provide any working example of an AED3-1 genomic knockout or edited AED3-1 plant , does not identify any AED3-1 guide RBA or target site, does not demonstrate any insertion, deletion, or substitution in AED3-1, and does not show that a plant or plant cell having such genomic modification actually enhances production of a heterologous protein. Thus, practicing the full scope of the genomic-modification embodiments would require undue experimentation.
Claims 4 and 15 recite AED3-1 comprising SEQ ID NO:1 or an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 1. The specification does not provide representative AED3-1 proteins across this broad 60% identity range or evidence that proteins having only 60% identity to SEQ ID NO: 1 retain AED3-1 activity or would function similarly in the claimed method. Because substantial sequence variation may affect protease activity, localization, substrate specificity, and susceptibility to inhibition, the disclosure does not enable the full scope of AED3-1 variants encompassed by the claims.
Claims 19-20 recites a nucleic acid molecule comprising SEQ ID NO: 2 or a reverse complement thereof, or a functional variant thereof. The specification sates that some variability within an shRNA sequence can be tolerated, for example 1, 2, or 3 nucleotide substitutions. However, the specification does not provide sufficient structural or functional guidance for determining which variants outside the specifically disclosed shRNA sequence would still inhibit AED3-1 and enhance heterologous protein production. Accordingly, the “functional variant” scope is broader than he enabling disclosure.
Therefore, although the specification enables the specific demonstrated shRNA-mediated inhibition of AED3-1 in N. benthamiana for trastuzumab production, the specification does not enable the full scope of claims 4-7, 12, and 15-20, including broad AED3-1 sequence variants, broad inhibitor classes, genomic modification embodiments, and functional variants of SEQ ID NO: 2, without undue experimentation.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 1-3, 8-9, 11-14, 16, and 18 are rejected under 35 U.S.C. §103 as being unpatentable over Grosse-Holz (Friederike Grosse-Holz et. al., Plant Biotechnology Journal (2018) 16, pp. 1797–1810), in view of (Stéphanie Robert et. al., PLOS ONE (2013) Volume8, Issue7, pp1-9) and Breitenbach (Heiko H Breitenbach et. al., Plant Physiology (2014) 22:165(2):791–809)
Claim 1 recites a method of enhancing production of a heterologous protein in a plant comprising: a) a nucleic acid molecule encoding the heterologous protein operably linked to a promoter; b) inhibiting aspartic protease AED3-1; and c) the plant expresses the heterologous protein.
Under the broadest reasonable interpretation as discussed above, consistent with the specification, “AED3-1” is not limited to the particular Nicotiana benthamiana sequence exemplified in the specification, but encompasses AED3/AED3-like apoplastic aspartic protease, including orthologous or corresponding AED3 proteins identified in other plant species.
Grosse-Holz teaches a method of enhancing production of heterologous recombinant proteins in Nicotiana benthamiana by agroinfiltration (p1797, Abstract). Grosse-Holz teaches introducing nucleic acid molecules encoding heterologous recombinant proteins, including α-galactosidase, erythropoietin, and the VRC01 antibody, into N. benthamiana leaves using Agrobacterium, and growing the infiltrated plant tissue to express the recombinant protein (Abstract; p1805, left and right column, “Cloning of PIs and RPs”). Grosse-Holz further teaches that recombinant protein accumulation is hampered by plant proteases and that co-expression of protease inhibitors, such as SICYS8 (cysteine protease inhibitor), NbPot1 (serine protease inhibitor), HsTIMP (metalloprotease inhibitor), can inhibit endogenous protease activity and enhances accumulation of recombinant protein (Abstract, Fig. 1).
Grosse-Holz does not expressly teach inhibiting aspartic protease AED3-1.
Robert teaches that endogenous proteolysis is a determinant of recombinant-antibody stability and yield in agroinfiltrated N. benthamiana and characterizes both cysteine-protease and aspartate protease activities during production of a recombinant antibody targeted to the plant secretory pathway (Abstract). Robert teaches that aspartate (A1) protease activity is present in whole-leaf and apoplastic extracts and that a substantial fraction of the detected aspartic-protease activity is located in the apoplast. Robert further teaches that agroinfiltration and heterologous-protein expression alter the extracellular protease environment (p4, left column, pa1). Thus, Robert identifies endogenous apoplastic aspartic protease as a protease class present in the same extracellular environment through which secreted recombinant proteins pass and in which proteolytic loss may occur.
Breitenbach teaches AED3 as a eukaryotic aspartyl-protease-family protein associated with protein metabolism and detects AED3 in an apoplast-enriched protein fraction (p796, Table 1; p795, right column, pa1). Breitenbach further teaches that AED family/apoplectic proteins participate in plant protein-processing and defense-related pathways (Abstract, Table 1).
It would have been obvious to a person of ordinary skill in the art, in view of Grosse-Holz, Robert and Breitenbach, to select AED3 for targeted inhibition in the recombinant-protein production method of Grosse-Holz. Grosse-Holz teaches that endogenous proteases limit recombinant-protein accumulation and that inhibition of selected endogenous proteases can improve accumulation. Robert teaches that apoplastic aspartate-protease activity, including activity attributed to A1 aspartate protease, is associated with proteolysis during recombinant-antibody production in N. benthamiana. Robert does not identify AED3 as the responsible protease. Breitenbach identifies AED3 as an endogenous apoplastic aspartyl-protease-family protein. Thus, Robert provides a reason to target members of the apoplastic aspartic-protease class, while Breitenbach identifies AED3 as a member of that relevant class.
The claim does not require a finding that AED3-1 was known to be the sole or predominant protease responsible for degradation of foreign proteins. Rather, the prior art identified a recognized problem “extracellular proteolysis of recombinant proteins” a relevant protease class-apoplastic aspartic protease- and a specific endogenous member of that class-AED3. A person of ordinary skill would therefore have had reason to test targeted inhibition of AED3 as one of a technically relevant apoplastic aspartic-protease candidates.
In view of these teachings, a person of ordinary skill in the art would have had reason to evaluate AED3 for target inhibition because Breitenbach identified AED3 as an endogenous apoplastic aspartyl protease belonging to the same protease class that Robert associated with recombinant-protein proteolysis.
Accordingly, claim 1 is obvious over Grosse-Holz in view of Robert and Breitenbach.
Claim 2 recites the method of claim 1, wherein step a) further comprises introducing into the plant or plant cell a nucleic acid molecule encoding a suppressor of gene silencing protein operably linked to a promoter, optionally P19.
For the same reason set forth above with respect to claim 1, Grosse-Holz teaches a method of enhancing production of a heterologous protein in N. benthamiana. Grosse-Holz further teaches co-expressing a suppressor of gene silencing, namely P19, with the heterologous protein to prevent the decline of transgene transcript levels and thereby increase recombinant protein accumulation (p1801,Fig 3; p1797, left column).
Accordingly, claim 2 is obvious over Grosse-Holz in view of Robert and Breitenbach.
Claim 3 recites the method of claim 1, wherein the plant or plant cell is of genus Nicotiana, optionally Nicotiana benthamiana.
For the same reason set forth above with respect to claim 1, Grosse-Holz teaches a method of enhancing production of a heterologous protein in N. benthamiana.
Accordingly, claim 3 is obvious over Grosse-Holz in view of Robert and Breitenbach.
Claim 8 recites the method of claim 1, wherein the heterologous protein is: a biologic drug; an antibody, optionally a human antibody or a humanized antibody; trastuzumab; insulin; a growth hormone; or a vaccine.
For the same reason set forth above with respect to claim 1, Grosse-Holz further teaches that the heterologous protein is a biopharmaceutical/recombinant proteins, including erythropoietin (EPO) and the IgG antibody VRC01 (p1797, Summary).
Accordingly, claim 8 is obvious over Grosse-Holz in view of Robert and Breitenbach.
Claim 9 method of claim 1, wherein the introducing into the plant comprises agroinfiltration, optionally with Agrobacterium tumefaciens (A. tumefaciens).
For the same reason set forth above with respect to claim 1, Grosse-Holz teaches introducing the heterologous protein expression construct into the plant by agroinfiltration with Agrobacterium tumefaciens.(p1797, introduction).
Accordingly, claim 9 is obvious over Grosse-Holz in view of Robert and Breitenbach.
Claim 11 recites the method of claim 9, wherein the heterologous protein is harvested less than about 7 days post-infiltration (dpi) or at about 3 dpi.
For the same reason set forth above with respect to claim 1, Grosse-Holz teaches that recombinant proteins were harvested/detected at 3 days post-infiltration (3 dpi) (p1798, right column, pa2).
Accordingly, claim 11 is obvious over Grosse-Holz in view of Robert and Breitenbach.
Claim 12 recites a modified plant with enhanced production of a heterologous protein, comprising: a) a nucleic acid molecule encoding linked to a promoter; and b) at least one of the group consisting of: i) an inhibitor of aspartic protease AED3-1; and ii) at least one genomic modification in a gene encoding aspartic protease AED3-1 conferring reduced or loss of function of the AED3- 1 as compared to a plant of the same species lacking the at least one genomic modification.
For the same reason set forth above with respect to claim 1, Grosse-Holz teaches a Nicotiana benthamiana plant or plant cell comprising a nucleic acid molecule encoding a heterologous recombinant protein operably lined to a promoter (Abstract and Fig 1, 2). Grosse-Holz further teaches that recombinant protein accumulation is improved by inhibiting plant protease activity (Abstract and Fig 1-2).
Breitenbach teaches, that AED3 is an endogenous apoplastic aspartyl protease associated with plant defense/protein metabolism.
Ite would have been obvious to prepare the modified plant or plant dell of claim 12 because it is the direct product/result of performing the obvious method of claim 1, namely introducing a heterologous protein expression cassette into the plant or plant cell and inhibiting AED3-1 to enhance heterologous protein production.
Accordingly, claim 12 is obvious over Grosse-Holz in view of Robert and Breitenbach.
Claim 13 recites the modified plant or plant cell of claim 12, further comprising a nucleic acid molecule encoding a suppressor of gene silencing protein operably linked to a promoter, optionally P19. Claim 14 recites the modified plant or plant cell of claim 12, wherein the plant or plant cell is of genus Nicotiana, optionally Nicotiana benthamiana (N. benthamiana). Claim 18 recites the modified plant or plant cell of claim 12, wherein the heterologous protein is: a biologic drug; an antibody, optionally a human antibody or humanized antibody; trastuzumab; insulin; a growth hormone; or a vaccine.
For the same reason set forth above with respect to claims 2, 3, 8, and 12, claims 13-14 and 18 are obvious over Grosse-Holz in view of Robert and Breitenbach.
Claim 16 recites the modified plant or plant cell of claim 12, wherein the inhibitor of AED3-1 comprises: a polypeptide; a small molecule drug; a shRNA, a siRNA, a miRNA, or an ASO; and/or a nucleotide comprising a nucleic acid sequence according to SEQ ID NO: 2 or a functional variant thereof.
For the same reason set forth above with respect to claim 12, Grosse-Holz teaches that the inhibitor may be a polypeptide protease inhibitor, including NbPR4, NbPot1, HsTIMP and SlCYS8, which enhance recombinant protein accumulation in agroinfiltrated Nicotiana benthamiana (p1997-1998, Abstract and Introduction). Because claim 6 recites the inhibitor alternatives using “and/or”, the polypeptide inhibitor limitation is sufficient.
Therefore, claim 16 would have been obvious over Grosse-Holz in view of Robert and Breitenbach.
Claims 4-6 and 15 are rejected under 35 U.S.C. §103 as being unpatentable over Grosse-Holz (2018), in view of Robert (2013), Breitenbach (2014) as apply to claims 1 and 12, and further in view of Sierro (Nicolas Sierro et. al., Nature Communications (2014) 5:3833, pp1-9).
Claims 1 and 12 as the teachings of Grosse-Holz, Robert and Breitenbach are discussed above.
Claims 4-5 are interpreted as dependent of claim 1.
Claim 15 is interpreted as dependent of claim 12.
Claim 4 recites the method of claim 1, wherein the AED3-1 comprises i) an amino acid sequence according to SEQ ID NO: 1 or ii) an amino acid sequence with at least 60% sequence identity to SEQ ID NO: 1. Claim 15 recites the modified plant or plant cell of claim 12, wherein the AED3-1 comprises i) an amino acid sequence according to SEQ ID NO: 1 or ii) an amino acid sequence with at least about 60% sequence identity to SEQ ID NO: 1.
For the same reason set forth above with respect to claims 1 and 12, Robert and Breitenbach provide reason to select AED3 for inhibition. Sierro teaches the tobacco genome sequence and provides the AED3/LOC107806163 sequence (below). The AED3 sequence is represented by RefSeq XP_016485758.1/ XM_016630272 and A0A1S4BAC6_TOBAC, as an AED3 protein, thereby providing both the sequence and its predicated AED3 function. Sequence comparison shows that SEQ ID NO: 1 is 100% identical to the AED3 sequence disclosed by Sierro (alignment see below).
Accordingly, a person of ordinary skill in the art would have identified this sequence as the tobacco AED3target for the inhibition taught by the combined reference. Claims 4 and 15 are obvious over Grosse-Holz in view of Robert, Breitenbach and Sierro.
Claim 5 recites the method of claim 1, wherein the inhibiting in b) comprises contacting the plant or plant cell with an inhibitor of AED3-1.
Under broadest reasonable interpretation, “contacting the plant or plant cell with an inhibitor of AED3-1” encompasses introducing the inhibitor into the plant by any suitable technique, including Agrobacterium-mediated transient expression (agroinfiltration).
For the same reason set forth above with respect to claims 1, Grosse-Holz teaches introducing proteinase inhibitors into Nicotiana benthamiana leaves by Agrobacterium-mediated agroinfiltration, whereby the inhibitor is delivered into and expressed within the infiltrated plant cells (p1806, left column, pa2). Therefore, Grosse-Holz teaches the claimed contacting step. Sierro teaches that AED3-1 (LOC107806613) is corresponding to the claimed AED3-1 sequence, as discussed above.
Accordingly, claim 5 would have been obvious over Grosse-Holz in view of Robert, Breitenbach and Sierro.
Claim 6 recites the method of claim 5, wherein the inhibitor comprises: a polypeptide; a small molecule drug; a short hairpin RNA (shRNA), a small interfering RNA (siRNA), a micro-RNA (miRNA), or an antisense oligonucleotide (ASO); and/or a nucleotide comprising a nucleic acid sequence according to SEQ ID NO: 2 or a functional variant thereof.
For the same reason set forth above with respect to claims 1 and 5, Grosse-Holz teaches that the inhibitor may be a polypeptide protease inhibitor, including NbPR4, NbPot1, HsTIMP and SlCYS8, which enhance recombinant protein accumulation in agroinfiltrated Nicotiana benthamiana (p1997-1998, Abstract and Introduction). Because claim 6 recites the inhibitor alternatives using “and/or”, the polypeptide inhibitor limitation is sufficient.
Therefore, claim 6 would have been obvious over Grosse-Holz in view of Robert, Breitenbach and Sierro.
Claims 7 and 17 are rejected under 35 U.S.C. §103 as being unpatentable over Grosse-Holz (2018), in view of Robert (2013), Breitenbach (2014) as apply to claims 1 and 12, and further in view of Sierro (2014) and Nekrasov (Vladimir Nekrasov et. al., Nature Biotechnology (2013) volume 31, pages691–693).
Claims 1 and 12 as the teachings of Grosse-Holz, Robert, and Breitenbach are discussed above. Claims 7 is interpreted as dependent of claim 1.
Claims 17 are interpreted as dependent of claim 12.
Claim 7 recites the method of claim 1, wherein the inhibiting in b) comprises: the inhibitor of AED3-1 operably linked to a promoter; and/or genomic modification in a gene encoding the AED3-1, confers reduced or loss of function of the AED3-1.
For the same reason set forth above with respect to claims 1, Grosse-Holz teaches enhancing recombinant protein accumulation in Nicotiana benthamiana by inhibiting endogenous plant protease through co-expression of protease inhibitors. Grosse-Holz teaches the cassette for protease inhibitors containing a 35S promoter and terminator (Fig 1C).
Grosse-Holz does not expressly teach introducing at least one genomic modification in a gene encoding AED3-1.
Sierro teaches the N. benthamiana AED3-1 sequence, which is 100% identical to SEQ ID NO: 1 of the instant application.
Breitenbach teaches AED3 is an apoplastic aspartyl protease family protein.
Nekrasov teaches CRISPR/Cas targeted mutagenesis in Nicotiana benthamiana using Agrobacterium-mediated delivery, producing targeted insertions and deletions at the selected gene locus (p691, right column pa2 to p692, left column and middle column).
It would have been obvious to introduce a genomic modification into AED3-1 to reduce or eliminate its function because Grosse-Holz teaches that reducing plant protease activity improves recombinant protein accumulation, Sierro identifies the AED3-1 target, Breitenbach identifies AED3 as an aspartyl protease, and Nekrasov teaches CRISPR/Cas as a routine method for generating targeted loss-of-function mutations in Nicotiana benthamiana.
Therefore, claim 7 would have been obvious over Grosse-Holz in view of Robert, Breitenbach, Sierro and Nekrasov.
Claim 17 recites the modified plant or plant cell of claim 12, wherein the at least one genomic modification conferring reduced or loss of function comprises an insertion, a deletion, or a substitution.
For the same reason set forth above with respect to claims 7 and 12, Nekrasov expressly teaches CRISPR/Cas9-induced targeted genomic modifications comprising insertions and deletions in Nicotiana benthamiana (p691, right column pa2 to p692, left column and middle column; Fig 1C).
Therefore, it would have been obvious for the genomic modification conferring reduced or loss of function of AED3-1 to comprise an insertion or deletion.
Accordingly claim 17 would have been obvious over Grosse-Holz in view of Robert, Breitenbach, Sierro and Nekrasov.
Claim 10 is rejected under 35 U.S.C. §103 as being unpatentable over Grosse-Holz (2018), in view of Robert (2013), and Breitenbach (2014) as apply to claims 1, and further review of Leth (Ingrid K. Leth et. al., Applied Microbiology and Biotechnology (2017) 101:4895–4903).
Claim 1 as the teachings of Grosse-Holz, Robert and Breitenbach are discussed above.
Claim 10 is interpreted as dependent of claim 1.
For the same reason set forth above with respect to claim 1, Grosse-Holz teaches large-scale agroinfiltration-mediated recombinant protein production in Nicotiana benthamiana (p1797, left column).
Grosse-Holz does not expressly teach growing Agrobacterium tumefaciens in a bioreactor prior to agroinfiltration.
Leth teaches that Agrobacterium-mediated transient expression is suitable for large-scale recombinant protein production in plants, and that production of Agrobacterium tumefaciens was scaled from shaking flasks to a 5-Lbenchtop reactor and further to a 100-L reactor to obtain sufficient bacterial culture for large-scale plant infiltration (Abstract).
It would have been obvious to grow Agrobacterium tumefaciens in a bioreactor prior to agroinfiltration because large-scale agroinfiltration requires large quantities of bacterial culture, and scaling Agrobacterium production from shake flasks to bioreactors was taught as a routine and predictable method for producing sufficient bacterial inoculum for plan infiltration.
Accordingly, claim 10 would have been obvious over Grosse-Holz in view of Robert, Breitenbach and Leth.
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432
975
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474
657
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Conclusion
No claims are allowed.
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/YANXIN SHEN/Examiner, Art Unit 1663
/WEIHUA FAN/Primary Examiner, Art Unit 1663
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