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 .
Election/Restrictions
Applicants’ election of Group I, Claims 1-17 and 19, without traverse, in the reply filed August 7, 2026, is acknowledged. Claim 18 has been canceled.
Claim Status
Claims 1-17 and 19-29 are pending. Claims 1-17, 19-20, and 25 have been amended. Claim 18 has been canceled. Claims 1-17 and 19 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 6 and 9-16rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 6 recites that the RNA m6A demethylase is “an engineered ALKBH5 or an engineered ALKBH5 homolog”. The specification identities numerous proteins as ALKBH5 homologs, including Arabidopsis ALKBH9B and ALKBH10B, rice Os9B, and ALKBH5 homologs from rapeseed, tobacco, alfalfa, sorghum, maize, wheat, and other plant species. The specification further states that plant ALKBH5 homologs ALKBH9B and ALKBH10B function as RNA m6A demethylases and that plant ALKBH5 homologs possess catalytic domains conserved with mammalian ALKBH5.
However, the specification does not provide an objective criterion defining the metes and bounds of the term “ALKBH5 homolog”. In particular, the specification does not state whether a protein qualifies as an ALKBH5 homolog based on a required degree of overall sequence identity, identity within the catalytic domain, phylogenetic relationship, conservation of particular structural features, RNA m6A demethylase activity, or some combination thereof. Although the specification separately recited embodiments having at least about 70% sequence identity to SEQ ID NO: 42-44, that sequence-identity limitation is not incorporated into claim 6 and does not expressly define the term “ALKBH5 homolog”.
Accordingly, one of ordinary skill in the art cannot determine with reasonable certainty which proteins fall within or outside the claimed genus of “ALKBH5 homologs”.
Dependent claims 9-16 are included in this rejection because they do not include additional limitations to resolve the ambiguity.
Claim 8 recites that “the endogenous region leads to assembly of the endogenous RNA m6A demethylase in foci or condensates within the cell”. The specification provides evidence that certain regions of the ALKBH5 are associated with condensate formation. For example, the specification reports that the C-termina region of ALKBH5 promotes phase separation and that ALKBH5 Δ298-394 did not form nuclear condensates.
However, the specification does not provide an objective standard for determining when an endogenous region “least to assembly” in foci or condensates. It is unclear whether the recited region must be necessary for assembly, sufficient for assembly, merely promote or increase assembly, or otherwise contribute to assembly. The claim also does not recite a measurable threshold or assay result by which one of ordinary skill could determine whether a particular endogenous region satisfies the limitation.
Accordingly, the phrase “leads to assembly of the endogenous RNA m6A demethylase in foci or condensates within the cell” does not provide reasonably certain boundaries for determining which endogenous regions fall within the scope of claim 8.
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.
Written Descriptions
Claims 1-8, 11-17, and 19 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
The Federal Circuit has clarified the application of the written description requirement. The court stated that a written description of an invention "requires a precise definition, such as by structure, formula, [or] chemical name, of the claimed subject matter sufficient to distinguish it from other materials". University of California v. Eli Lilly and Co., 119 F.3d 1559, 1568; 43 USPQ2d 1398, 1406 (Fed. Cir. 1997). The court also concluded that "naming a type of material generally known to exist, in the absence of knowledge as to what that material consists of, is not description of that material". Id. Further, the court held that to adequately describe a claimed genus, Patent Owner must describe a representative number of the species of the claimed genus, and that one of skill in the art should be able to "visualize or recognize the identity of the members of the genus". Id.
Claim 1 broadly encompasses a DNA molecule encoding an engineered RNA m6A demethylase having at least one disrupted endogenous LCR and/or IDR for expression in a plant or plant cell. Claim 6 further encompasses an engineered ALKBH5 homolog, Claim 7 encompasses proteins having as little as about 70% sequence identity to SEQ ID NO: 42-44, and claim 11 encompasses an engineered plant ALKBH5 homologs, but continue to depend from claim 1 and therefore encompasses disruption of an LCR/IDR generally unless a particular disruption is expressly required. Claims 12-15 identify particular plant ALKBH5-family members; however, the recited deletion regions are expressly optional. Accordingly, these claims continue to encompass disruption of LCR/IDR regions generally through their dependency from claims 11 and 1 and are not limited to the particular LCR/IDR disruptions demonstrated in the specification
The specification provides working disclosure for a substantially narrower set of proteins and modification. For human ALKBH5, the specification tests ALKBH5 Δ30-81, ALKBH5 Δ298-394, and ALKBH5 Δ30-81/298-394 (pa0464-0473). For plant proteins, the specification testes selected engineered forms of Arabidopsis ALKBH9B, rice Os9B, and rice Os10B, including ALKBH9B Δ432-507, Os9B Δ428-616, and Os10B Δ491-595 (pa0511-0515). Thus, the disclosure demonstrates possession of particular ALKBH5-family members having particular identified LCR/IDR disruptions.
The specification itself also demonstrates that these structural regions and homologs are not functionally interchangeable. ALKBH5 Δ298-394, and ALKBH5 Δ30-81/298-394 promoted Arabidopsis root growth, whereas ALKBH5 Δ30-81 had only a limited effect (pa0465). Likewise, the specification reports that Arabidopsis ALKBH9B exhibited greater activity than ALKBH10B in the assay used the inventors (pa0512). The specification further acknowledges that relevant plant m6A demethylase exhibit major differences outside the catalytic domain, precisely where the claimed LCR/IDR disruptions occur (pa0446-0447).
The state of the art similarly demonstrates that specific structural features, rather than general family membership, govern ALKBH5-family function. Feng (Chong Feng et. al., Journal of Biological Chemistry (2014) Vol 289(17), pp11571-11583) determined that ALKBH5 contains structural features distinct from other AlkB proteins and that a particular “lid” region plays a vital role in substrate recognition and catalysis (p11571, left column, pa1); Feng further identified specific residues important for selective substrate binding (p11575, left column, pa1). Thus, conservation of the general AlkB catalytic fold does not itself establish equivalent substrate-recognition properties throughout the broader family.
Martinez-Perez (Mireya Martínez-Pérez et. al., PNAS (2017) vol 114 (40), pp 10755–10760) likewise shows functional specialization among related proteins. Martinez-Perez demonstrated specifically that Arabidopsis ALKBH9B removes m6A from single-stranded RNA and additionally reported its localization in cytoplasmic granules (p10755, left column, pa1). Subsequent work (Martínez-Pérez et. al., Frontiers in Microbiology (2021) 12:745576, pp1-13) showed that related Arabidopsis ALKBH9A and ALKBH9C did not perform the same role as ALKBH9B in the tested AMV infection system, with ALKBH9B activity being described as highly specific (Abstract). This evidence shows that close family relationship does not establish common biological properties across related ALKBH proteins.
Further Alvarado-Marchena (Luis Alvarado-Marchena et. al., Frontiers in Plant Science (2021) vol 12, 701683, pp1-12) mapped functional subdomain of ALKBH9B and found that particular C-terminal residues were critical for RNA binding, other residues were critical for protein interaction, and deletion of terminal regions altered accumulation cellular bodies (Abstract). Qin (Xiaoyang Qin et. al., JBC (2023) 299(8) 105071, pp1-12) similarly demonstrated that the intrinsically disordered C-terminal region of human ALKBH5 has a specific biological role in phase separation and localization (Title; Abstract). These references reinforce that IDRs/LCRs may contain specific functional determinants and cannot be treated simply as interchangeable non-catalytic sequence.
Against this evidence, the claims extend to any RNA m6A demethylase having a disrupted LCR/IDR, an undermined ALKBH5-homolog genus, proteins having as little as 70% sequence identity to the identified sequences, and broad plant-homolog groups without requiring the particular regions demonstrated to be operative.
Accordingly, while the specification reasonably demonstrates possession of particular engineered ALKBH5 proteins and selected plant homologs containing particular LCR/IDR disruptions, the specification does not provide a representative number of species or common structural characteristic sufficient to demonstrate possession of the full scope of claims 1-8, 11-17, and 19.
Scope of Enablement
Claims 1-8, 11-17, and 19 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 the specifically disclosed engineered RNA m6A demethylases, including human ALKBH5, Arabidopsis ALKBH9B and ALKBH10B, and rice Os9B and Os10B, and the specific LCR/IDR deletions exemplified for those proteins; does not reasonably provide enablement for the full scope of the claimed invention, including the broader genus of RNA m6A demethylases and ALKBH5 homologs having any disrupted endogenous LCR and/or IDR. 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.
Claim 1 encompasses any engineered RNA m6A demethylase having at least one disrupted endogenous LCR and/or IDR for expression in a plant or plant cell. Claim 6 further encompasses an engineered ALKBH5 homolog, Claim 7 encompasses proteins having as little as about 70% sequence identity to SEQ ID NO: 42-44, and claim 11 encompasses an engineered plant ALKBH5 homologs. Claims 12-15 identify particular plant ALKBH5-family members; however, the recited deletion regions are expressly optional.
The specification provides adequate techniques for identifying candidate LCRs and IDRs. For example, the disclosure uses IUPred with a score threshold exceeding 0.5 together with AlphaFold structural predictions to identify disordered regions and design truncations (pa0445). Thus, the enablement deficiency is not merely how to identify an LCR/IDR, but rather how to predict which demethylase, which LCR/IDR, and which disruption will produce an operative engineered RNA m6A demethylase across the full claimed scope.
The specification demonstrates that selected embodiments can be made and tested. Human ALKBH5Δ30-81, ALKBH5 Δ298-394, and ALKBH5 Δ30-81/298-394 were constructed and evaluated, and selected IDR-deleted Arabidopsis ALKBH9B, rice Os9B, and rice Os10B variants were likewise tested (pa0464-0473, 0511-0515).
However, the specification’s own experimental results demonstrate unpredictability. ALKBH5 Δ298-394 and the double deletion promoted Arabidopsis root growth, whereas ALKBH5Δ30-81 had only a limited effect (pa0465). Among related Arabidopsis proteins,, ALKBH9B and ALKBH10B also showed different activity in the inventors’ assay (pa0512). The disclosure therefore does not establish that identification and disruption of an LCR/IDR alone predictably produce equivalent functional results.
Feng confirms the same structure-function unpredictability (p11575, left column, pa1). Feng showed that ALKBH5 substrate recognition and catalysis depend on particular structural elements, including a distinct lid region and specific residues included in substrate recognition (p11571, left column, pa1). Thus, retention of general catalytic-domain relationship does not make the effect of sequence alteration elsewhere in the protein predictable.
Similarly, Martinez-Perez (2017) established RNA m6A demethylase activity for the particular plant protein ALKBH9B, rather than establishing equivalent activity across all related ALKBH proteins(p10755, left column, pa1) . Later work Martinez-Perez (2021) specifically found that related Arabidopsis ALKBH9A and ALKBH9C did not regulate the same biological process as ALKBH9B, demonstrating functional specialization among closely related family members (Abstract) .
Alvarado-Marchena also demonstrates that the very disordered regions targeted by the claims can themselves perform specific functions. Alvarado-Marchena found that defined regions of the ALKBH9B C-terminus are important for RNA binding, protein interaction, and localization, and that terminal deletions alter accumulation in cellular bodies (Abstract). Qin likewise showed that the disordered C-terminus of ALKBH5 mediated phase separation and that deletion of this region alters ALKBH5-associated cellular functions (Abstract and Title). Accordingly, an LCR/IDR cannot be presumed to be an expendable region whose disruption will predictably retain the desired demethylase properties.
The specification does not provide a generally applicable structure-function rule that resolved this unpredictability. To practice the full claimed scope, one of ordinary skill would therefor need to identify candidate RNA m6A demethylases or ALKBH5 homologs; identify one or more candidate LCR/IDR in each protein; select particular regions and expends of disruption; construct the resulting variants; and experimentally determine whether each engineered protein retains the required RNA m6A demethylase function. Nonoperative or functionally altered variants would have to be discarded and additional variants designed and tested.
The breadth of the claims is substantial; the working examples are concentrated on human ALKBH5 and selected Arabidopsis/rice homologs and selected deletion regions; both the specification and the state of the art demonstrate functional differences among homologs and among different IDR/LCR regions; and the disclosure lacks predictive structure-function guidance commensurate with the full claimed scope. Although the individual molecular-biological assays and engineering techniques are known, applying them throughout the claimed genus would require substantial empirical screening rather than routine verification of predictably operative embodiments.
Claim Rejections - 35 USC § 102
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.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 1, 3- 8, 11, 12, and 19 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Alvarado-Marchena (Luis Alvarado-Marchena et. al., Frontiers in Plant Science (2021) vol 12, 701683, pp1-12).
Claim 1 recites a DNA molecule encoding an engineered RNA m6A demethylase, comprises at least one disrupted endogenous region, which is a low complexity region (LCR) and/or intrinsically disordered region (IDR), and wherein the DNA molecule is operably linked to a promoter for expression in a plant or plant cell.
Alvarado-Marchena discloses the Arabidopsis thaliana atALKBH9B as an RNA m6A demethylase and further discloses engineered deletion mutants thereof (Abstract). Alvarado-Marchena expressly reports that 45.4% of the atALKBH9B amino-acid sequence forms IDRs located in the N- and C- terminal regions (p4, left column, pa1), and Figure 4 identifies the disordered residues in the atALKBH9B amino-acid sequence (Abstract; p6, Fig.4). Alvarado-Marchena further discloses engineered atALKBH9B mutants comprising deletions within IDR region of the protein (p9, left column, pa3; Fig 2 and 6), thereby disclosing recombinant DNA encoding an engineered RNA m6A demethylase having a disrupted endogenous IDR.
Claim 3 recites the DNA molecule of claim 1, wherein the RNA m6A demethylase comprises a disrupted endogenous N-terminal region and/or a disrupted C- terminal region.
Alvarado-Marchena discloses disruption of the N-terminal and/or C-terminal regions of atALKBH9B. in particular, Alvarado-Marchena discloses Δ160Nt and Δ160Ct mutants and additional C-terminal deletion mutants, and separately examines deletion of the first N-terminal 20 residues and the last C-terminal 40 residues (p5, right column, pa3; P3, left column, pa3; Fig 2A; Fig 1A; Fig. 6).
Claim 4 recites the DNA molecule of claim 1, wherein the disruption of the endogenous region is selected from the group consisting of a partial truncation, a full truncation, a deletion, and a replacement.
Alvarado-Marchena discloses the deletions/truncations, including Δ160Nt, Δ160Ct, Δ258Nt, Δ80Ct, Δ40Ct, Δ20Ct, Δ20Nt, and other deletion constructs (Fig. 2A, 6A-B).
Claim 5 recites the DNA molecule of claim 1, wherein the RNA m6A demethylase comprises a deletion of at least about 10% to 100% of an endogenous region, wherein the region is an LCR or IDR.
Alvarado-Marchena discloses deletion of portions of endogenous regions identified as IDRs. Alvarado-Marchena expressly states that the deleted N-terminal 20 residues and C-terminal last 40 residues are predicted to form IDRs (Abstract; Fig. 4B, 6B). Alvarado-Marchena identifies a C-terminal IDR spanning approximately amino acids 437-507 (71 amino acids) (Fig 7). Thus, the deletion removes 56.3% of an endogenous IDR as required by claim 5.
Claim 6 recites the DNA molecule of claim 1, wherein the RNA m6A demethylase is an engineered ALKBH5 or an engineered ALKBH5 homolog.
Alvarado-Marchena discloses engineered atALKBH9B, a plant AlkB-family RNA m6A demethylase corresponding to the claimed plant ALKBH5-milolog embodiment. Alvarado-Marchena states that atALKBH9B has m6A-demethylase activity and identifies it among the Arabidopsis AlkB homologs (Abstract and Introduction).
Claim 7 recites the DNA molecule of claim 1, wherein the RNA m6A demethylase comprises an amino acid sequence with at least about 70% to 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 42-44.
Alvarado-Marchena expressly discloses the amino acid sequence of the Arabidopsis thaliana RNA m6A demethylase atALKBH9B (Fig. 4A). Alvarado-Marchena identifies the AlkB-like domain of atALKBH9B as located at amino acids 216-411 (Fig 7; p6, right column). Sequence alignment of SEQ ID NO: 43 with the atALKBH9B sequence shows that SEQ ID NO: 43 is identical to amino acids 296-419 of atALKBH9B, with 124/124 amino acids identical (100%) and no gape (see below alignment).
Claim 8 recites the DNA molecule of claim 1, wherein the endogenous region leads to assembly of the endogenous RNA m6A demethylase in foci or condensates within the cell.
Alvarado-Marchena discloses that atALKBH9B forms discrete cytoplasmic granules and colocalized with SGS3-containing siRNA bodies, described as biomolecular condensates associated with stress granules (p5, right column, pa1). Alvarado-Marchena further shows that deletion of the N-terminal 20 residues or C-terminal 40 residues, which are predicted IDRs, prevents such accumulation (p5, right column pa1-3), thereby demonstrating that these endogenous regions are involved in granule/condensates assembly.
Claim 11 recites the DNA molecule of claim 6, wherein the RNA m6A demethylase is an engineered plant ALKBH5 homolog.
For the same reason set forth above with respect to claim 6, Alvarado-Marchena discloses an engineered plant ALKBH homolog, namely Arabidopsis thaliana atALKBH9B, in the form of the above deletion mutants (Abstract, Fig. 2 and 6).
Claim 12 recites the DNA molecule of claim 11, wherein the RNA m6A demethylase is Arabidopsis ALKBH9B, optionally wherein the ALKBH9B comprises a deletion of at least about 10% to 100% of:(i) amino acids 76-102,(ii) amino acids 145-183, and/or (iii) amino acids 432-507 of endogenous ALKBH9B (SEQ ID NO: 3).
For the same reason set forth above with respect to claim 11, Alvarado-Marchena discloses Arabidopsis thaliana atALKBH9B and provides the complete atALKBH9B amino-acid sequences (Fig. 4A). Sequence alignment of the atALKB9B sequence disclosed in Figure 4A with SEQ ID NO: 3 of the instant application shous 507/507 amino acids identical, i.e., 100% identity with no gaps (see alignment below). Alvarado-Marchena additionally discloses deletion of IDR-containing N-and C-terminal regions of this same atALKBH9B protein (Fig. 2A, 4A, and 6A-B).
Claim 19 recites an expression vector comprising the DNA molecule of claim 1.
1, 3-6, 8, 11, 12, and 19 discloses expression vectors comprising DNA encoding the engineered atALKBH9B deletion mutants. Specifically, the full-length atALKBH9B ORF and deletion mutants were subcloned into pGEX-KG and expressed as GST:atALKBH9B fusion proteins (p9, left column, pa3).
Accordingly, claims 1, 3-8, 11, 12, and 19 are anticipated by 1, 3-6, 8, 11, 12, and 19.
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Claims 1, 6, 9, 10, and 17 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Fang (Ruiqiu Fang et. al., Plant Methods (2023 )19(1):81, pp1-8).
Regarding claim 1, Fang discloses a DNA molecule encoding an engineered RNA m6A demethylase comprising the human ALKBH5 catalytic domain, amino acids 66-292, fuse within a plant m6A editor (Abstract; p2, left column, pa2). Sequence alignment of the ALKBH5 portion encoded by Fang’s expression cassette with SEQ ID NO: 1 of the instant application shows 100% amino-acid identity over residues 66-292 of SEQ ID NO: 1 (see below).
Fang further discloses expression of the ALKBH5-containing construct under a CMV 35S promoter and transformation and expression of the construct in Arabidopsis (see supplementary “Additional file 1 Sequence of dlwCas13a-msfGFP-ALKBH cassette”; Fig. 1A-B). Fang demonstrates that engineered construct decreases m6A modification of targeted RNAs, thereby confirming RNA m6A demethylase activity (Abstract).
Fang’s ALKBH5 aa 66-292 construct necessarily lacks endogenous portion of full-length ALKBH5 outside aa66-292, including the endogenous C-terminal region. Qin (2023) provides evidence that human ALKBH5 comprises a catalytic domain at approximately aa 74-294 and that the flanking N- and C-terminal regions are intrinsically disordered regions (IDRs) (p2, left column, pa4). Qin further demonstrates that the C-terminal IDR is responsible for ALKBH5 phase separation (Title). Qin is relied upon as extrinsic evidence establishing the inherent structural characteristic of the ALKBH5 construct expressly disclosed by Fang, rather than as a second reference supplying a missing limitation.
Accordingly, Fang inherently discloses an engineered RNA m6A demethylase comprising a disrupted endogenous IDR.
Regarding claim 6, for the same reason set forth above with respect to claim 1, Fang discloses that the engineered RNA m6A demethylase comprises human ALKBH5, specifically the ALKBH5 catalytic domain aa66-292.
Claim 9 recites the DNA molecule of claim 6, wherein the engineered RNA m6A demethylase is an engineered ALKBH5 comprising a deletion of at least about 10-100% of:(i) amino acids 30-81 and/or (ii) amino acids 298-394 of endogenous ALKBH5 (SEQ ID NO: 1).
For the same reason set forth above with respect to claim 1, Fang’s ALKBH5 aa 66-292 construct corresponds to residues 66-292 of SEQ ID NO: 1 and therefore necessarily lacks the claimed endogenous ALKBH5 C-terminal region corresponding to amino acids 298-394. Thus, Fang discloses deletion of 98% of the recited aa 298-394 region, satisfying the claimed deletion of at least 10%-100% of that region.
Claim 10 recites the DNA of claim 9, wherein the RNA m6A demethylase is selected from the group consisting of ALKBH5Δ30-81, ALKBH5Δ298-394, and ALKBH5Δ30-81 and 298-394.
For the same reason set forth above with respect to claim 1, 6, and 9, Fang discloses an engineered human ALKBH5 construct comprising the ALKBH5 catalytic domain corresponding to amino acids 66-292 of endogenous ALKBH5 (p2, left column, pa2; Fig. 1A; Supplementary “Additional file 1 Sequence of dlwCas13a-msfGFP-ALKBH cassette”). The disclosed construct therefore lacks the endogenous ALKBH5 region comprising amino acids 298-394. Accordingly, Fang discloses an engineered ALKBH5 having the deletion recited as ALKBH5Δ298-394, as required by claim 10.
Claim 17 recites the DNA molecule of claim 1, wherein the RNA m6A demethylase is operably linked to at least one nuclear localization signal (NLS).
For the same reason set forth above with respect to claim 1, Fang discloses addition of nuclear localization signal (NLS) peptides, including an NLS at the C-terminus of the ALKBH5-containing fusion protein (p2, lest column, pa2).
Accordingly, claims 1, 6, 9, 10, and 17 are anticipated by Fang.
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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.
Claim 13 is rejected under 35 U.S.C. §103 as being unpatentable over Alvarado-Marchena (2021) as apply to claim 11, and in view of Duan (Hong-Chao Duan et. al., The Plant Cell (2017) Vol. 29: 2995–3011).
Claim 11 as the teachings of Alvarado-Marchena are discussed above.
Claim 13 is interpreted as dependent of claim 11.
Claim 13 recites the DNA molecule of claim 11, wherein the RNA m6A demethylase is Arabidopsis ALKBH1OB (SEQ ID NO: 5).
For the same reason set forth above with respect to claim 11, Alvarado-Marchena teaches that the related Arabidopsis RNA m6A demethylase atALKBH9B contains endogenous IDRs and teaches engineered deletion mutants comprising deletion within such IDRs and teaches engineered deletion mutants comprising deletion within such IDRs.
Duan teaches Arabidopsis thaliana ALKBH10B (At4g02940), an RNA m6A demethylase (p2996, left column, pa2). Sequence alignment of the ALKBH10B (At4g02940) with SEQ ID NO: 5 of the instant application demonstrates 100% sequence identity. Thus, Duan teaches the Arabidopsis ALKBH10B corresponding to SEQ ID NO: 5 as recited in claim 13.
It would have been obvious to one of ordinary skill in the art to apply the IDR-disruption approach taught by Alvarado-Marchena to Duan’s Arabidopsis ALKBH10B to provide an engineered plant RNA m6A demethylase comprising a disrupted endogenous IDR, with a reasonable expectation of obtaining an engineered ALKBH-family demethylase having an altered disordered region.
Claim 13 is obvious over Alvarado-Marchena and Duan.
Claims 14-16 are rejected under 35 U.S.C. §103 as being unpatentable over Alvarado-Marchena (2021) as apply to claim 11, in view of Zhao (Ye Zhao et. al., Frontier in Plant Science (2022) 19, pp1-15).
Claim 11 as the teachings of Alvarado-Marchena are discussed above.
Claim 14-16 are interpreted as dependent of claim 11.
Claim 14 recites the DNA molecule of claim 11, wherein the RNA m6A demethylase is Os9B (SEQ ID NO: 6). Claim 15 recites DNA molecule of claim 11, wherein the RNA m6A demethylase is Os10B (SEQ ID NO: 8).
For the same reason set forth above with respect to claim 11, Alvarado-Marchena teaches that the related Arabidopsis RNA m6A demethylase atALKBH9B contains endogenous IDRs and teaches engineered deletion mutants comprising deletion within such IDRs and teaches engineered deletion mutants comprising deletion within such IDRs.
Zhao teaches a rice ALKBH9-family protein identified as Os06g0138200 in the ALKBH9 phylogenetic group (Fig. 2). Rice Genome Hub identifies Os06g0138200 as LOC_Os06g04660.1, and sequence alignment of LOC_Os06g04660.1 with SEQ ID NO: 6 of the instant application demonstrates 100% sequence identity (alignment see below). Thus, Zhao teaches the rice Os9B protein corresponding to SEQ ID NO: 6 as recited in claim 14.
Zhao teaches a rice ALKBH10-family protein identified as Os10g0116900 in the ALKBH10 phylogenetic group (Fig. 2). Sequence alignment of Os10g0116900 with SEQ ID NO: 8 of the instant application demonstrates 100% sequence identity (alignment see below). Thus, Zhao teaches the rice Os10B protein corresponding to SEQ ID NO: 8 as recited in claim 15.
It would have been obvious to one of ordinary skill in the art to apply the IDR-disruption approach taught by Alvarado-Marchena to Zhao’s rice ALKBH9-family and ALKBH10-family to provide an engineered plant RNA m6A demethylase comprising a disrupted endogenous IDR, with a reasonable expectation of obtaining an engineered ALKBH-family demethylase having an altered disordered region.
Claims 14-15 are obvious over Alvarado-Marchena and Zhao.
Claim 16 recites the DNA molecule of claim 11, wherein the RNA m6A demethylase is selected from the group consisting of rapeseed ALKBH9B, tobacco ALKBH9B, tobacco ALKBH10B, alfalfa ALKBH9B, sorghum ALKBH9B, maize ALKBH9B, maize ALKBH10B, wheat ALKBH9B, and wheat ALKBH10B.
For the same reason set forth above with respect to claim 11, Zhao teaches ALKBH-family proteins from maize and wheat that are classified within the ALKBH9 and ALKBH10 phylogenetic groups (Fig.2). Thus, Zhao teaches at least one of the plant ALKBH9B/ALKBH10B alternatives recited in claim 16.
Alvarado-Marchena teaches engineering a related plant ALKBH m6A demethylase by disruption of an endogenous IDR. It would have been obvious to apply the IDR-disruption approach of Alvarado-Marchena to the maize or wheat ALKBH9/ALKBH10 homologs taught by Zhao.
Claim 16 is obvious over Alvarado-Marchena and Zhao.
Claim 2 is rejected under 35 U.S.C. §103 as being unpatentable over Fang (2023) as apply to claim 1, and in view of Qin (2023).
Claim 1 as the teachings of Fang are discussed above.
Claim 2 is interpreted as dependent of claim 1.
Claim 2 recites the DNA molecule of claim 1, wherein the at least one disrupted endogenous region is a low complexity region (LCR).
For the same reason set forth above with respect to claim 1, Qin teaches that intrinsically disordered regions are characterized by low sequence complexity and specifically identify the C-terminal region of ALKBH5 as an intrinsically disordered region (p2, right column, pa2).
It would have been obvious to one of ordinary skill in the art to characterize/select the disrupted disordered region taught by Fang as a low-complexity region because Qin teaches low sequence complexity as a characteristic of such IDRs.
Claim 2 is obvious over Fang and qin.
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716
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878
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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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