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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on February 13, 2026 has been entered.
Claim 1 is amended. Claims 1 and 4-5 remain pending and are examined on their merit herein.
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 and 4-5 remain 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.
Claim 1 is rejected as being indefinite for the following reasons:
Claim 1 is amended to recite a “method of replacing an undesired polynucleotide that encodes an endogenous gene comprising an allele responsible for disease susceptibility of the NLB 18 gene at its native location within a genomic region between NLB17 and NLB18, wherein the genomic region comprises repetitive regions of other NLB polynucleotide sequences within a maize plant”. It is unclear what the scope of the claim is.
The claim is drawn to a method of “replacing an undesired polynucleotide”. The “undesired polynucleotide” is defined as a “polynucleotide that encodes an endogenous gene”; In turn, the “endogenous gene” is limited by “comprising an allele responsible for disease susceptibility of the NLB 18 gene at its native location within a genomic region between NLB17 and NLB18”. However, it is not clear whether the “endogenous gene” is an allele of NLB18, or some unspecified gene in “a genomic region between NLB17 and NLB18”.
The Specification defines the term “allele” as:
[0035] An “allele” is one of several alternative forms of a gene occupying a given locus on a chromosome. When all the alleles present at a given locus on a chromosome are the same, that plant is homozygous at that locus. If the alleles present at a given locus on a chromosome differ, that plant is heterozygous at that locus.
By this definition, the recited “an allele …… of the NLB18 gene” should be interpreted as “occupying” the same locus of the “NLB18 gene”. In other words, the recited “endogenous gene” is a different allele (version) of the “NLB18 gene”.
However, the claim also appears to define the “endogenous gene” as something located “between NLB17 and NLB18”.
Furthermore, if the endogenous gene is interpreted as being located “between NLB17 and NLB18”, then it is not clear what is the “allele responsible for disease susceptibility”, since the allele is not at the same locus of NLB18.
It is therefore not clear whether the “undesired sequence” is the NLB18 allele that is responsible for disease susceptibility, or some undefined sequence “between NLB17 and NLB18”.
Therefore, the metes and bounds of the claim are not clear.
Claims 4-5 are included in this rejection for their failure to correct the deficiency above.
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.
Claims 1, and 4-5 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.
Neither the instant specification nor the originally filed claims appear to provide support for the limitation “an endogenous gene comprising an allele responsible for disease susceptibility of the NLB 18 gene at its native location within a genomic region between NLB17 and NLB18”.
The amendment to claim introduces the new matter of an endogenous gene …… within a genomic region between NLB17 and NLB18 in the claimed method of removing such a gene. The Specification has described the method of removing the endogenous NLB18 gene from a susceptible maize cultivar, via a pair of gRNAs hybridizing to the promoter region and of the 3′ UTR of the NLB18-S allele. ([0246] “NLB18-S deletion was achieved by introducing two guide RNAs (gRNAs) to generate two cuts in the DNA of maize inbred line PH1V5T in predetermined locations: one 5′ guide RNA homologous to the upstream sequence located in the promoter region of the susceptible allele, and a 3′ guide RNA homologous to the downstream sequence located in the 3′ UTR of the susceptible allele”). However, the Specification has not described any deletion of the “endogenous gene” located “between NLB17 and NLB18” and wherein the endogenous gene comprises an allele responsible for disease susceptibility, since this allele will not be NLB18 allele.
Thus, such a limitation constitutes NEW MATTER. In response to this rejection, Applicant is required to point to support for the limitation or to cancel the new matter.
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 and 4-5 remain rejected under 35 U.S.C. 103 as being unpatentable over Elison et al (Elison et al. Cell reports 18.1 (2017): 275-286) in view of Gao et al (WO2018071362A1; Published 19 Apr 2018).
Due to Applicant’s amendment of the claims, the rejection is modified from the rejection set forth in the Office action mailed November 19th, 2025. Applicant’s arguments regarding the rejection have been fully considered, see response at the end of the rejection.
AS discussed above, the amendment to Claim 1 is introduced the limitation to define the “undesired polynucleotide” as a “polynucleotide that encodes an endogenous gene comprising an allele responsible for disease susceptibility of the NLB 18 gene at its native location within a genomic region between NLB17 and NLB18”.
Claim interpretation:
As discussed above, the claim is indefinite because it is unclear whether the undesired polynucleotide sequence is allelic to NLB18—i.e., “occupying” the same genomic locus of NLB18, or something unspecified “between NLB17 and NLB18”.
Furthermore, it should be noted that the maize NLB 18 locus, as taught in the instant Specification, is an exemplary of such a genomic region with “highly repetitive sequences”.
For the purpose of this rejection, the undesired polynucleotide is interpreted as polynucleotide that encodes an endogenous gene comprising an allele responsible for disease susceptibility of the NLB 18 gene. The limitation of “between NLB17 and NLB18” is not taken into consideration since it is conflicting with rest of the claim.
In this claimed method, the sequences flanking the region excised by the Cas endonuclease above are brought closer together to form a third target sequence, which is then targeted by a third guide RNA provided to the cells concurrently with a heterologous polynucleotide having at least 95% identity to the originally excised sequence, which is also flanked by regions having homology to the sequences flanking the originally excised sequence. In this claimed method, the heterologous sequence is then inserted at the site of the newly formed target sequences, effectively replacing the undesired sequence that was excised in the steps above by the Cas endonuclease. This will be referred to as the “two-step gene swapping method”.
Claim 4 is further drawn to introducing a second Cas endonuclease in Step (b) of the method above. Claim 5 is further drawn to the method of claim 1, wherein the cell in step (b) of the method is a progeny of a cell in step (a).
Teachings of the prior art:
Elison et al discloses a two-step method for replacing polynucleotide sequences using CRISPR-Cas effector systems in yeast (Abstract; Pg. 275, right col, ¶3).
In the first step of their disclosed method, Elison et al discloses the use of a pair of guide RNAs that hybridize to regions flanking an undesired polynucleotide sequence contained within a promoter operably linked to a nucleic acid encoding yellow fluorescent protein (YFP) (Pg. 285, left col, ¶4; Pg. 276, ¶2-5). A Cas endonuclease is provided to the cells which, through the targeting by the guide RNAs, cleaves the two regions that flank the undesired polynucleotide sequence (Pg. 276, ¶2-5; Figure 1). An oligonucleotide concurrently provided to the organism which contains regions with homology to the regions flanking the undesired polynucleotide targeted by the guide RNAs is then used to guide the repair of the cleaved polynucleotide in the cell, effectivity replacing the undesired polynucleotide with the sequence of this oligonucleotide—known as the “donor oligonucleotide” (Pg. 276, ¶2-8; Figure 1).
The sequence supplied by Elison et al on the donor oligonucleotide that replaced the originally excised polynucleotide was short (30bp) and contained a new target sequence amendable to cleavage by a Cas endonuclease (Pg. 276, ¶3; Figure 1). Thus, the result of this first method step disclosed by Elison et al is the removal of an undesired polynucleotide through the action of a Cas endonuclease, which brings the sequences flanking the undesired polynucleotide closer together and forms a third target sequence between said flanking sequences (Figure 1).
In the second step of their disclosed method, Elison et al discloses the use of a third guide RNA targeting the newly formed third target sequence from the first step (Pg. 276, ¶3-4; Figure 1). A similar donor oligonucleotide is provided as above which contains a modified version of the originally excised promoter sequence (Pg. 276, ¶3-4; Figure 1). As with the first method, Elison et al discloses that the donor oligonucleotide containing the modified version of the originally excised polynucleotide is flanked by regions that share identity with the regions flanking the originally targeted sequence (Figure 1). Through DNA repair mechanisms, the sequence of this donor oligonucleotide is integrated into the site of the double-stranded break initiated by a Cas endonuclease at the newly created site targeted by the third guide RNA (Figure 1).
Elison et al discloses that the result of the second step of their method is the integration of the modified version of the originally excised polynucleotide contained within the second donor polynucleotide (Pg. 276, ¶2-5; Figure 1). Elison et al discloses several examples of polynucleotides used to replace the original, undesired promoter sequence—including those with only a single binding site for a cis-acting element removed (GE13; 97.5% identity to the excised sequence) and those with rearrangements, modifications, and insertions (Table S1; III. Supplemental Experimental Procedures). The polynucleotides being swapped by Elison et al were contained within a promoter region responsive to galactose operably linked to YFP; the substitutions disclosed by Elison et al conferred varying levels of fluorescence in yeast cells in response to galactose—a desired phenotype which furnished an understanding of the properties of the promoter elements of the GAL1 and GAL80 promoters in yeast (Figures 2-6; Pg. 284, ¶2-5).
In summary, Elison et al discloses a two-step gene swap method with a first and second step analogous to steps (a) and (b) of the methods set forth in claims 1 and 3. In the first step of the gene replacement method above, Elison et al discloses the cleavage of two flanking sequences with a Cas endonuclease targeted by a pair of guide RNAs, which results in said flanking sequences being brought closer together and results in the formation of a third target sequence. In the second step of their disclosed method, Elison et al discloses that the newly formed target sequence is targeted by a third guide RNA, cleaved by a Cas endonuclease, then repaired in a manner directed by a concurrently provided oligonucleotide containing a sequence with high identity to the originally excised sequence and flanked by regions sharing high homology to sequences flanking to originally excised polynucleotide. The result of this second step of their disclosed method is the replacement of the original, undesired polynucleotide with a similar polynucleotide which confers a desirable phenotype to the organism (Figures 1-6; Pg. 284, ¶2-5).
Figure 1 from Elison et al summarizes this two-step gene swapping procedure. Figure 1A and Figure 1B from Elison et al are included below, with panel A describing the first step and panel B describing the second step of their disclosed method:
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In addition to the disclosed gene swapping method performed in yeast, Elison et al teach that their gene swapping method can be applied to other organisms (Pg. 284, ¶3). In addition, Elison et al teach that their disclosed method has several advantages over other single step gene swapping methods known in the art—particularly the lack of any lingering polynucleotide sequences adjacent to the undesired polynucleotide (called “scars”) that may impact the function of the inserted polynucleotide (Pg. 275, ¶2-3; Pg. 284, ¶2). Because of the flexibility of their two-step method, Elison et al teach that their method could be applied to directed evolution experiments and analyses of structure and function of gene networks in other biological systems (Pg. 284, ¶2-3; Abstract).
However, Elison et al do not specifically teach the application of their disclosed gene swapping method to plants. By extension, Elison et al do not specifically teach the application of their disclosed gene swapping method to maize. Elison et al also does not teach an NLB18-S polynucleotide or an NLB18-R polynucleotide.
Gao et al teach methods for obtaining plant cells with modified NLB18 sequences, including methods which involve introducing double-strand breaks into the maize genome in an endogenous NLB18 encoding sequence to modify the genomic sequence in order to enhance northern leaf blight resistance of a plant produced from the plant cell (Abstract).
Gao et al teaches that CRISPR-Cas endonuclease systems may be used to replace alleles of gene involved in disease resistance with alleles which confer resistance to a specific disease or pathogen (Pg. 34 lines 1-26; Figure 5). Gao et al teaches that NLB18 is a locus on chromosome 8 in maize which determines resistance to northern leaf blight (Pg. 42 lines 11-19). Gao et al teaches that the NLB18 locus corresponds to a gene encoding a wall-associated kinase; Gao et al also teaches that CRISPR-Cas effector systems may be used to replace NLB18 alleles associated with susceptibility to northern leaf blight may be replaced with alleles which confer tolerance to northern leaf blight (Pg. 59 lines 1-16). Gao et al provides a specific example of maize plants produced with such a system, specifically in which the NLB-TS4 allele was replaced with an NLB-CR4 allele which conferred resistance to northern leaf blight (Pg. 57 line 2 – Pg. 58 line 21; Tables 9-10; Figure 5).
Gao et al teach specific polynucleotide sequences associated with the wall-associated kinase (SEQ ID NOs: 30-35; Tables 9-10). Gao et al also teach that the gene swapping methods of their disclosure, which resemble those of Elison et al above, are amenable to excising and replacing genes in repetitive regions of the maize genome (Pg. 52 Line 11 – Pg. 54 Line 8).
Furthermore, regarding the limitation “the genome region comprising repetitive regions of polynucleotide sequences”, the Specification has not provided any direct definition of this term. In relation, the Specification defines “genome region” as the following:
[0022] As used herein, a "genomic region" is a segment of a chromosome in the genome
of a cell that is present on either side of the target site.
It is noted that the above cited definition does not limit the size of the "genomic region", as far as it is a segment of a chromosome, and flanking the target site. Read in light of this, it is interpreted that “the genome region comprising repetitive regions of polynucleotide sequences” is the segment of a chromosome surrounding the target site, and in this segment there are repetitive sequences.
In the case of Gao and the “NLB18” target, it is noted that the NLB18 sequence (i.e., the target site, which is marked by the cDNA sequence SEQ ID NO: 61) is mapped to a segment of maize chromosome 8. As captured in the genome browser annotation below (from https://jbrowse.maizegdb.org/), any 10 kb, 20 kb, …… 100 kb, or longer, “segments” of the chromosome comprising the target site (marked by “BLAST hit”) comprise a number of repetitive sequences (transposable elements and sirevirus LTR retrotransposons).
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Gao teaches replacing an undesired NLB18 allele with a desired one: (p. 4, lines 3-18:
“The methods may further comprise introducing an
NLB18 substitution template in the maize plant cell, wherein said NLB18
substitution template comprises at least one nucleic acid alteration
compared to the endogenous NLB18 encoding sequence and wherein
said NLB18 substitution template is incorporated into the endogenous
NLB18 encoding sequence. The double-strand break may be induced by
a nuclease such as but not limited to a TALEN, a meganuclease, a zinc
finger nuclease, or a CRISPR-associated nuclease. The method may
further comprise growing a maize plant from the maize plant cell having
the modified NLB18 nucleotide sequence, and the maize plant may exhibit
enhanced resistance to northern leaf blight.”
Furthermore, Gao even teaches replacing the susceptible NL18 allele with the one from the resistant genotype:
an NLB18 subsitution template is used, which
comprises an NLB18 nucleotide sequence from PH26N or PH99N
(NLB18-PH26N or NLB18-PH99N). (p. 4, lines 27-29).
It should be noted that the working example used in the instant disclosure, the replacement NLB18 allele is also from maize cultivar PH26N (Instant Specification, [0246]).
Therefore, Gao already taught the method of replacing an undesired polynucleotide, i.e., an NLB18 from susceptible maize cultivar, with an NLB18 allele from a resistant maize cultivar, via CRISPR-mediated genomic editing technology.
At the time the invention was filed, one of ordinary skill in the art would be motivated to apply the gene swapping method disclosed by Elison et al to substitute a maize NLB18 allele conferring susceptibility to northern leaf blight for an NLB18 allele conferring resistance to northern leaf blight to maize, as taught by Gao et al. One of ordinary skill in the art would be motivated to do so to increase the yield of maize subjected to disease stress from northern leaf blight. Furthermore, one of ordinary skill in the art would be motivated to develop such methods to generate novel allelic variation in maize that can be integrated into maize breeding programs.
CRISPR-Cas gene editing systems have been extensively examined in several biological systems, given the ability of such effector systems to make precise edits in the genome of several organisms. Accordingly, Gao et al teaches that modifications to DNA sequences—including alterations, insertions, and gene swaps—using CRISPR-Cas systems have been evaluated in maize (Pg. 16 Line 4 – Pg. 30 line 19). In addition, Elison et al teaches that their invention is applicable to several other organisms, and specifically provides guidance for how their method may be modified to adapt said method to other organismal systems (specifically animal systems) when an essential gene is excised in the first step (Pg. 284, ¶2-4). Given these teachings, one of ordinary skill in the art would be motivated to apply the gene swapping method taught by Elison et al to maize, as taught by Gao et al.
As diseases in maize, including northern leaf blight, can lead to crop failure in maize (Gao et al Pg. 1 Lines 19-30), one of ordinary skill in the art would be motivated to apply methods known in the art at the time of filing to confer resistance to northern leaf blight. One of ordinary skill in the art would recognize that the NLB18 alleles which are associated with susceptibility to northern leaf blight in maize could be substituted for an NLB18 allele which confers resistance to northern leaf blight, including the NLB-CR4 allele taught by Gao et al (SEQ ID NO:35; Table 10).
As Gao et al teach that a gene swapping procedure using a CRISPR-Cas effector system was applied to successfully replace NLB18 alleles in a similar manner, one of ordinary skill in the art would be able to arrive at such a method with a reasonable expectation of success.
Thus, one of ordinary skill in the art would arrive at the claimed gene swapping method of claim 1, in which an NLB18 allele conferring susceptibility to northern leaf blight is replaced with an allele conferring resistance to northern leaf blight (understood to be encompassed by NLB18-S and NLB18-R, respectively). Since the replacement allele would be in a different genome context, it would be technically “heterologous”. (Specification, [0057]: “The term "heterologous" refers to the difference between the original environment, location, or composition of a particular polynucleotide or polypeptide sequence and its current environment, location, or composition.) This, one or ordinary skill in the art would arrive at the invention set forth in the instant claim 1.
With respect to claim 4, the use of different Cas endonucleases is made obvious to one of ordinary skill in the art because Gao et al teaches several different Cas endonucleases which are applicable to the compositions and methods of their invention, including Cpf1, C2c1, C2c2, C2c3, Cas3, Cas 5, Cas7, and Cas10 (Pg. 14 Lines 12-26).
With respect to claim 5, use of progeny is made obvious to one of ordinary skill in the art because Gao et al teaches that progeny of the plants of their disclosure are encompassed, and because Gao et al teaches that the gene swapping methods of their disclosure resulted in stable transformants (Pg. 19 Line 27 – Pg. 20 Line 7; Pg. 59 Lines 1-28).
Therefore, the claimed invention as a whole is prima facie obvious over the combined teachings of the prior art.
Response to Applicant’s Remarks:
Applicant’s argument that Elison required the incorporation of a donor oligonucleotide. Which is not taught suggested or motivated by instant diclosure’s third target sequence "that is complementary to the sequence of the repaired second double strand break polynucleotide".
This argument has been fully considered but not deemed persuasive.
Firstly, the instantly claimed method, at the second step, requires a donor nucleotide. Secondly, Gao provided the teaching and motivation of using the donor NLB18 from a resistant maize genotype to replace the NLB18 in susceptible maize lines—exactly the same strategy of the instant disclosure. Therefore, the combined teachings of the prior art renders the instant claims prima facie obvious.
Applicant further argued that neither Elison nor Gao teaches or suggests that a single-step approach would fail in plants, nor that success would require sequential regeneration and retransformation. This argument has been fully considered but not deemed persuasive.
Elison, as discussed above, has taught the advantage of the two step method for its precisiveness. Since PHOSITA would have been motivated to adopt this two-step method, the failure of single-step approach is irrelevant.
Therefore, at least for these reasons, the rejection is maintained.
Conclusion
No claims are allowed.
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WEIHUA . FAN
Primary Examiner
Art Unit 1663
/WEIHUA FAN/Primary Examiner, Art Unit 1663