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 .
Applicant’s response of 11/24/2025, including amendments to the specification, has been received and entered into the application file.
Claims 1, 4, 5, and 9 were amended in the claim set filed 11/24/2025.
Claims 2, 3, and 6-8 were cancelled in the claim set filed 11/24/2025.
Claim 10 was added in the claim set filed 11/24/2025.
Accordingly, claims 1, 4, 5, 9, and 10 are pending and under consideration.
Status of Prior Objections/Rejections
RE: Specification
►The specification was previously objected to for a number of informalities.
The amendments to the specification received on 11/24/2025 have obviated the basis of the prior objections. The objections of record are hereby withdrawn.
RE: Claim Objections
►Claims 1-9 were previously objected to for minor informalities.
The cancellation of claims 2, 3, and 6-8 renders the objections thereof moot.
The amendments to the instant claim set have obviated the basis of the prior objections. The objections of record are hereby withdrawn. New grounds of objection are set forth below.
RE: Claim Rejections - 35 USC § 112(b)
►Claims 5 and 8 were previously 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.
The cancellation of claims 5 and 8 renders the rejections thereof moot.
RE: Claim Rejections - 35 USC § 112(d)
►Claim 2 was previously rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
The cancellation of claim 2 renders the rejection thereof moot.
RE: Claim Rejections - 35 USC § 102
►Claims 1, 2, and 9 were previously rejected under 35 U.S.C. 102(a)(1) as being anticipated by Mandal et al., 2014.
The cancellation of claim 2 renders the rejection thereof moot.
Applicant has traversed the rejection of record, asserting that Mandal et al., 2014 does not teach or suggest one or more elements of amended instant claim 1.
In response, this is found persuasive. The rejection of record is hereby withdrawn. However, the amendments to the instant claim set have necessitated new grounds of rejection, as set forth below.
RE: Claim Rejections - 35 USC § 103
►Claims 3-7 were previously rejected under 35 U.S.C. 103 as being unpatentable over Mandal et al., 2014 as applied to claim 1, and further in view of WO 2021/061707 A1 (hereinafter Omega), as evidenced by Baumann et al., 2002.
►Claim 8 was previously rejected under 35 U.S.C. 103 as being unpatentable over Mandal et al., 2014 and WO 2021/061707 A1 (hereinafter Omega), as evidenced by Baumann et al., 2002, as applied to claim 7, and further in view of Liu et al., 2017, Seki and Rutz, 2018, and Addgene #71489 (reported in Arbab et al., 2015).
The cancellation of claims 3 and 6-8 renders the rejections thereof moot.
Applicant has traversed the rejections of record, asserting that the Examiner combined references by utilizing hindsight reasoning, starting from Applicant’s solution of a specific vector ratio designed to optimize enrichment of co-transfected cells. Applicant further asserts that one of ordinary skill in the art would not look to the cited references for guidance, as the references address fundamentally different technical problems. Specifically, Applicant asserts that Seki (of Seki and Rutz, 2018) teaches excess gRNA over Cas9 for optimization of intracellular biochemical efficiency of the CRISPR system, while the instant application optimizes the intercellular statistical probability of enriching a cell population successfully receiving all required sgRNAs by adjusting the ratio between the drug-resistance vector and the sgRNA vectors.
In response, regarding the assertion of use of improper use of hindsight, the Examiner notes that only new instant claim 10 recites a specific vector ratio and that this claim was only added in the amendments filed 11/24/2025. Amended instant claims 1, 4, 5, and 9 do not recite any specific ratio, but rather they recite (or inherit the recitation of) “an amount of the vector containing the drug-resistance gene used in transfection is less than amount of each vector containing one sgRNA of the one or more sgRNA groups or each vector containing one or more of the sgRNA groups used in transfection.” While the Examiner acknowledges that the disclosure of Seki and Rutz, 2018 teaches that the gRNA vector is administered in excess of the Cas9 vector for purposes of increasing knockout efficiency rather than for optimization of intercellular statistical probability of enriching a cell population, as previously set forth, the combination of Liu et al., 2017, Addgene #71489, and Seki and Rtuz, 2018 discloses that in a system with multiple guide RNAs, each guide RNA vector is provided in excess of the Cas9 vector comprising a drug-resistance gene. In view of the language of the instant claim set, these vectors expressing Cas9 and comprising a drug-resistance gene are considered to read on the instantly claimed drug-resistance vector. Thus, this disclosure teaches administration of sgRNA vector(s) in excess of drug-resistance vectors, as instantly claimed.
The instant claim language does not recite that this ratio is for purposes of optimizing intercellular statistical probability of enriching a cell population. Furthermore, even if the instant claim language did recite that this ratio is for such purposes, per MPEP § 2114 and § 2173.05(g) when a prior art reference or a combination of prior art references disclose the claimed subject matter, the claimed and disclosed subject matter must be structurally identical and therefore must necessarily have identical functions, such as optimizing intercellular statistical probability of enriching a cell population, even if the prior art does not explicitly disclose the shared function.
Applicant further asserts that the claimed methods yield synergistic and unexpected results, achieving a greater efficiency in generating mutant embryonic stem cells with deleted target genes than typical methods of generating large deletions. Applicant asserts that the instantly claimed method achieves an efficiency of 31.25% in deleting target genes, while <1% efficiency is typically reported in the prior art for generating large deletions.
In response, the Examiner again notes that the instant claim language does not recite large deletions, and is instead drawn to deletion of target genes. Furthermore, Applicant’s assertion that the methods documented in the prior art have <1% efficiency in generating large deletions is not found credible. The term “large deletion” is not clearly defined in the instant specification. However, paragraph [0029] does disclose “mutant cells with a large deletion of more than 1000 bp,” while paragraphs [0021] and [0025] respectively disclose “mutant cells with deleted target genes” and “deletion of a large segment of the target gene.” Therefore, deletions of entire genes or segments of genes greater than 1000 bp are considered to constitute “large deletions” under broadest reasonable interpretation. In view of this interpretation, Eleveld et al., 2021 discloses successful deletions of 65 and 53 Mb using CRISPR-Cas9 in up to 30% of selected clones (abstract). Both of these deletions are greater than 1000 bp and certainly encompass deletion of target genes or large segments thereof. Accordingly, the disclosure of Eleveld et al., 2021 establishes that CRISPR-Cas9 is a robust genome editing technology that has much greater than <1% efficiency in generating deletion of target genes or large segments thereof. Thus, the results disclosed in the instant application are not considered to represent synergistic or unexpected results.
However, in view of the amendments to the instant claim set, the rejections of record are hereby withdrawn, while new grounds of rejection necessitated by amendment are set forth below.
New/Maintained Grounds of Objection/Rejection
Claim Objections
Claims 1, 4, and 10 are objected to because of the following informalities:
Claims 1, 4, and 10 each recite the acronym “sgRNA.” While those of ordinary skill in the art would generally be aware that “sgRNA” is an acronymization for “synthetic single-guide RNA” that combines the trans-activating CRISPR RNA (tracrRNA) and CRISPR RNA (crRNA) into a single RNA transcript (Jiang and Doudna, 2017: abstract; page 509, paragraph 2), it is nonetheless proper to recite the species that is being acronymized before reciting the acronym thereof. It would be remedial to amend the instant claim set such that “synthetic single-guide RNA” is explicitly recited prior to the first recitation of its associated acronym (i.e. at instant claim 1).
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, 4, 5, 9, and 10 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.
Claims 1, 4, 5, 9, and 10 are drawn to a method of preparing mutant cells with deleted target genes, said method comprising transfecting one or more sgRNA groups and a drug-resistance gene into host cells. The rejected claims thus comprise editing compositions for deleting target genes that comprise one or more sgRNA groups.
To provide adequate written description and evidence of possession of a claimed genus, the specification must provide sufficient distinguishing identifying characteristics of the genus. The factors to be considered include disclosure of a complete or partial structure, physical and/or chemical properties, functional characteristics, structure/function correlation, and any combination thereof. The specification describes editing compositions for deleting target genes that comprise two different sgRNAs (paragraph [0057]). These two sgRNAs satisfy the definition of an sgRNA group in the instant application (amended claim 1; paragraph [0006]). Thus, the specification discloses a method of preparing mutant cells with deleted target genes, said method comprising transfecting one sgRNA group and a drug-resistance gene into host cells. No description is provided of such a method comprising transfecting more than one sgRNA group, as encompassed by the range of the limitation “one or more,” which is recited at instant claim 1 and inherited by claims 4, 5, 9, and 10.
Even if one accepts that the examples described in the specification meet the claim limitations of the rejected claims with regard to structure and function, the examples are only representative of a method of preparing mutant cells with deleted target genes, said method comprising transfecting one sgRNA group and a drug-resistance gene into host cells. The results are not necessarily predictive of such a method in which an unlimited number of sgRNA groups are administered, as is encompassed by the limitation of “one or more.” Furthermore, the prior art does not appear to offset the deficiencies of the instant specification in that it does not describe methods of preparing mutant cells with deleted target genes in which an unlimited number of sgRNA groups are administered. While the utility of multiple sgRNAs in deleting large segments of genomic sequence is known in the art (Eleveld et al., 2021: abstract; Figure 1), the limitation of “one or more” necessarily encompasses up to an unlimited number of sgRNA groups, which is not supported by either the instant specification or by the prior art.
Therefore, the skilled artisan would have reasonably concluded applicants were not in possession of the claimed invention for claims 1, 4, 5, 9, and 10.
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 9 are rejected under 35 U.S.C. 103 as being unpatentable over Mandal et al., 2014 (of record) in view of WO 2021/061707 A1 (hereinafter Omega; of record), Liu et al., 2017 (of record), Seki and Rutz, 2018 (of record), and Addgene #71489 (reported in Arbab et al., 2015; of record), as evidenced by Jiang and Doudna, 2017 (of record).
With regard to amended instant claim 1, which recites “a method for preparing mutant cells with deleted target genes, comprising:
step (1): transfecting one or more sgRNA groups and a drug-resistance gene into host cells, wherein each of the one or more sgRNA groups includes two sgRNAs that target two different sites of a same target gene, and each different sgRNA groups targets one different target gene; wherein the one or more sgRNA groups and the drug-resistance gene are respectively included on different expression vectors; when the number of the sgRNA groups is more than one, the sgRNA groups are included on separate expression vectors or on a same expression vector; wherein two sgRNAs of a sgRNA group are included on separate expression vectors or on a same expression vector; wherein an amount of the vector containing the drug-resistance gene used in transfection is less than an amount of each vector containing one sgRNA of the one or more sgRNA groups or each vector containing one or more of the sgRNA groups used in transfection;
step (2): enriching the host cells expressing one or more sgRNA groups;
step (3): culturing the host cells obtained from step (2) to produce mutant cells with deleted target genes,” as previously set forth, Mandal et al., 2014 discloses a dual guide RNA approach to CRISPR/Cas9 editing in primary human CD4+ T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs) that improves gene deletion efficacy in both cell types (abstract). This dual gRNA approach targeting separate sites in separate genes B2M and CCR5 (with dual gRNAs targeting each gene) is shown in Figure 2 and is disclosed to facilitate predicted deletions rather than the small InDels produced with single gRNAs (page 650, column 1, paragraph 2). The disclosed methods include cell transfection with Cas9 and dual gRNAs (page 650, column 2, paragraph 2) followed by sorting and culturing cells to produce mutant cells with deleted target genes (supplemental information: page 3, paragraph 3-page 4, paragraph 2), as instantly claimed. The gRNAs of Mandal et al., 2014 are disclosed to be separately expressed from individual plasmids (supplemental data: section “Molecular Biology” of “EXPERIMENTAL PROCEDURES”). Thus, Mandal et al., 2014 discloses a method for preparing mutant cells, said method comprising transfecting Cas9 and two sgRNA groups, in which each sgRNA group comprises two sgRNAs that target two different sites of the same target gene wherein the sgRNAs are all included on separate expression vectors, followed by sorting and culturing cells to produce mutant cells with deleted target genes, as instantly claimed. However, Mandal et al., 2014 does not disclose the instantly claimed expression vector containing a drug-resistance gene, nor do they disclose that the amount of the vector containing the drug-resistance gene used in transfection is less than an amount of each vector containing one sgRNA of the one or more sgRNA groups or each vector containing one or more of the sgRNA groups used in transfection.
These deficiencies are cured by the various secondary references, as set forth below.
As previously set forth, Omega discloses agents and compositions for reducing apolipoprotein B (APOB) expression, including site-specific targeting moieties such as guide RNAs (also referred to as sgRNAs) that may be transfected into host cells via expression vectors (abstract; page 7, lines 23-35; page 20, lines 6-9; page 27, lines 5-10; page 37, lines 14-18; page 61, lines 2-12 and 27-29; Example 1). Omega further discloses that the expression vector(s) taught therein may also contain a selectable marker such as an antibiotic resistance gene to facilitate identification and selection of appropriately transfected cells (page 62, line 36-page 63, line 9). This selectable marker may also be delivered on a separate piece of DNA and used in a co-transfection procedure rather than delivered on the same expression vector carrying the guide RNA taught therein (page 62, line 36-page 63, line 9). This transfection of a drug-resistance gene to the host cells targeted for editing reads on the instantly claimed step of transfecting a drug-resistance gene into the host cells in which the sgRNA sand drug-resistance genes are included on different expression vectors. Thus, Omega discloses the instantly claimed expression vector containing a drug-resistance gene, teaching its role in successful detection of gene editing using CRISPR.
Furthermore, as set forth above, Omega discloses that the expression vector(s) taught therein may also contain a selectable marker such as an antibiotic resistance gene to facilitate identification and selection of appropriately transfected cells and that this selectable marker may also be delivered on a separate piece of DNA and used in a co-transfection procedure rather than delivered on the same expression vector carrying the guide RNA taught therein (page 62, line 36-page 63, line 9). While Omega does not disclose that these separate vectors are administered such that the amount of the vector containing the drug-resistance gene is lower than the amount of the vector containing each sgRNA, this deficiency is collectively cured by Liu et al., 2017, Seki and Rutz, 2018, and Addgene #71489 (reported in Arbab et al., 2015).
Liu et al., 2017 discloses that dual vectors can be used to deliver guide RNAs and Cas9 separately, both of which are required for successful genome editing, as the guide RNA is only capable of recognizing its target sequence in the genome, while the Cas9 nuclease acts as a pair of scissors to cleave the double strands of DNA (page 23, column 2, paragraph 5; abstract). Vectors expressing only guide RNAs and only Cas9 (or plasmids useful for building such vectors) are known to those of ordinary skill in the art. Mandal et al., 2014 explicitly discloses that the Cas9 and gRNAs utilized therein were expressed from separate plasmids (supplemental data: section “Molecular Biology” of “EXPERIMENTAL PROCEDURES”).
One such vector known in the art is Addgene #71489 (spCas9-BlastR; reported in Arbab et al., 2015), which expresses only Cas9 and utilizes blasticidin as a selectable marker to isolate successfully transfected cells. Thus, this vector reads on the instantly claimed vector containing a drug-resistance gene as recited at instant claim 1. Seki and Rutz, 2018 further disclose that knockout efficiency dramatically increases when a guide RNA is provided in excess of Cas9 (specifically a 3:1 excess ratio) (page 986, column 2, paragraph 2). Given that guide RNAs bind to their respective target sequences and only their respective target sequences and are not generally known to function cooperatively or to interfere with each other (reviewed in Jiang and Doudna, 2017), it is considered that Seki and Rutz, 2018 discloses that in a system with multiple guide RNAs (such as the dual guide RNA system disclosed in Mandal et al., 2014), each guide RNA must be provided in excess of Cas9 (contained within a vector such as Addgene #71489, which includes blasticidin as a drug-resistance gene to serve as a selectable marker to isolate successfully transfected cells) to increase knockout efficiency. As set forth above, this Cas9 vector reads on the instantly claimed vector containing a drug-resistance gene. Thus, these references collectively disclose that the amount of the vector containing the drug-resistance gene (and Cas9 per the cited references) used in transfection is less than an amount of each vector containing one sgRNA of the one or more sgRNA groups or each vector containing one or more of the sgRNA groups used in transfection, as instantly claimed.
With regard to amended instant claim 9, which recites “a mutant cell with deleted target genes prepared from the method according to claim 1,” not only does Mandal et al., 2014 disclose the dual gRNA approach set forth above, but they also disclose in vivo transplantation and single cell PCR assays of the edited cells (Figure 3; supplemental information: page 4, paragraph 3-page 5, paragraph 1), which inherently requires the claimed mutant cell with a deleted targeted gene, as instantly claimed.
Given that Mandal et al., 2014 discloses a dual guide RNA approach to CRISPR/Cas9 editing, that Omega discloses delivery of gene disrupting agents such as gRNAs via transfection of host cells with expression vectors comprising said gRNAs and/or drug-resistance genes to facilitate identification and selection of appropriately transfected cells, and that Liu et al., 2017, Addgene #71489 (spCas9-BlastR; reported in Arbab et al., 2015), and Seki and Rutz, 2018 collectively disclose that Cas9 may be delivered on a separate plasmid also comprising a drug-resistance gene (thereby reading on the instantly claimed vector containing a drug-resistance gene) at an amount that is less than the guide RNA vectors, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to deliver the dual guide RNA vectors (as disclosed in Mandal et al., 2014 and Omega) and Cas9 vector comprising a drug-resistance gene (such as Addgene #71489) separately (as disclosed in Liu et al., 2017), with each guide RNA vector in excess of the Cas9 vector comprising a drug-resistance gene (as disclosed in Seki and Rutz, 2018) to predictably increase knockout efficiency in the targeted cells. One would have been motivated to make such a modification in order to receive the expected benefit of increasing knockout efficiency in the targeted cells.
Claims 4 and 5 are rejected under 35 U.S.C. 103 as being unpatentable over Mandal et al., 2014 (of record) in view of WO 2021/061707 A1 (hereinafter Omega; of record), Liu et al., 2017 (of record), Seki and Rutz, 2018 (of record), and Addgene #71489 (reported in Arbab et al., 2015; of record), as evidenced by Jiang and Doudna, 2017 (of record), as applied to claim 1 above, and further in view of Baumann et al., 2002 (of record).
The combined disclosures of Mandal et al., 2014, Omega, Liu et al., 2017, Addgene #71489 (reported in Arbab et al., 2015), and Jiang and Doudna, 2017 are described above and applied as before. However, these disclosures do not teach the step of employing a drug to remove the host cells that do not contain the drug-resistance gene, specifically an antibiotic, as in instant claims 4 and 5.
With regard to amended instant claim 4, which recites “enriching the host cells expressing one or more sgRNA groups in the step (2) [of the method of claim 1] comprises employing a drug to remove the host cells that do not contain the drug-resistance gene,” as previously set forth, while Omega discloses transfection with drug-resistance genes, as instantly claimed and set forth above, to facilitate identification and selection of appropriately transfected cells (page 62, line 36-page 63, line 9), they are silent as to methods of using said drug-resistance gene for identification and selection of appropriately transfected cells. However, these methods are well-known to those of ordinary skill in the art. For example, Baumann et al., 2002 discloses a selectable gene marker (mcrA) suitable for use in mammalian cells that selects for transfected cells by either pulsing or continuously culturing the cells with the corresponding antibiotic to more efficiently isolate successfully transfected cells (mitomycin C) (abstract; page 4, column 2, paragraph 2 (underneath Figure 1)). This methodology of transfecting a drug-resistance gene into targeted cells followed by the addition of the same drug to kill cells that were not successfully transfected is well-known in the art, as evidenced by Baumann et al., 2002.
With regard to amended instant claim 5, which recites “the drug [of the method of claim 4] is selected from antibiotics,” as set forth above regarding instant claim 3, Omega discloses that the expression vector(s) taught therein to deliver APOB disrupting agents such as gRNAs may also contain a selectable marker such as an antibiotic resistance gene to facilitate identification and selection of appropriately transfected cells (abstract; page 7, lines 23-35; page 20, lines 6-9; page 27, lines 5-10; page 37, lines 14-18; page 61, lines 2-12 and 27-29; page 62, line 36-page 63, line 9; Example 1). Per Baumann et al., 2002, transfected cells may be selected by either pulsing or continuously culturing the cells with the antibiotic corresponding to the antibiotic resistance gene being utilized to more efficiently isolate successfully transfected cells (abstract; page 4, column 2, paragraph 2 (underneath Figure 1)).
Given that Mandal et al., 2014, Omega, Liu et al., 2017, Addgene #71489 (reported in Arbab et al., 2015), and Jiang and Doudna, 2017 collectively disclose the method of amended instant claim 1 as set forth above, and that Baumann et al., 2002 discloses that addition of the same drug corresponding to the transfected drug-resistance gene (i.e. an antibiotic) to the cell culture facilitates identification and selection of appropriately transfected cells, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of amended instant claim 1 (collectively disclosed as set forth above) to further comprise transfection with a drug-resistance gene (i.e. an antibiotic resistance gene) to predictably to facilitate identification and selection of appropriately transfected cells following addition of the same drug (i.e. an antibiotic) to the cell culture (as disclosed in Omega and Baumann et al., 2002). One would have been motivated to make such a modification in order to receive the expected benefit of identifying and selecting appropriately transfected cells that are thus more likely to be properly edited.
Claim 10 is rejected under 35 U.S.C. 103 as being unpatentable over Mandal et al., 2014 (of record) in view of WO 2021/061707 A1 (hereinafter Omega; of record), Liu et al., 2017 (of record), Seki and Rutz, 2018 (of record), and Addgene #71489 (reported in Arbab et al., 2015; of record), as evidenced by Jiang and Doudna, 2017 (of record), as applied to claim 1 above, and further in view of Eleveld et al., 2021.
The combined disclosures of Mandal et al., 2014, Omega, Liu et al., 2017, Addgene #71489 (reported in Arbab et al., 2015), and Jiang and Doudna, 2017 are described above and applied as before. However, these disclosures do not teach the ratio of the amount of the sgRNA and drug-resistance vectors of instant claim 10.
With regard to new instant claim 10, which recites “a ratio of the amount of the vector containing the drug-resistance gene [of the method of claim 1] used in transfection to a total amount of vectors containing each one sgRNA of the one or more sgRNA groups or vectors containing one or more sgRNA groups used in transfection is in a range of 1:20 to 1:4,” as set forth above regarding amended instant claim 9, Seki and Rutz, 2018 further disclose that knockout efficiency dramatically increases when a guide RNA is provided in excess of Cas9 (specifically a 3:1 excess ratio) (page 986, column 2, paragraph 2). Further, as set forth above, vectors expressing only Cas9 and comprising drug-resistance markers are known in the art (i.e. Addgene #71489) and read on the instantly claimed vector containing a drug-resistance gene as recited at instant claim 1. Thus, while it is considered the Seki and Rutz, 2018 (in combination with the secondary references) discloses that vectors containing sgRNA group(s) are provided in excess of the vectors containing the drug-resistance gene, they do not disclose the instantly claimed range of ratios.
Eleveld et al., 2021, also discloses administration of dual gRNAs targeting a single gene to generate large deletions (abstract; Figure 1). Eleveld et al., 2021 further discloses that the gRNAs taught therein were provided at a 6:1 ratio to Cas9 (page 12008, column 2, paragraph 2: “RNP complexes and transfection”). This ratio falls within the instantly claimed range of 1:20 to 1:4. Thus, the disclosures of both Seki and Rutz, 2018 and Eleveld et al., 2021 establish that it is within the realm of routine experimentation for someone of ordinary skill in the art to manipulate the administered ratio of guide RNAs to Cas9 (which is known to be included in vectors expressing only Cas9 and comprising drug-resistance markers, thereby reading on the instantly claimed vector containing a drug-resistance gene as recited at instant claim 1). Per MPEP § 2144.05(II)(A), “it is not inventive to discover the optimum or workable ranges by routine experimentation” (In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955)). Therefore, it is considered that the cited art motivates someone of ordinary skill in the art to establish optimum or workable ranges by routine experimentation, which is not inventive per MPEP § 2144.05(II)(A).
Given that Mandal et al., 2014, Omega, Liu et al., 2017, Addgene #71489 (reported in Arbab et al., 2015), and Jiang and Doudna, 2017 collectively disclose the method of amended instant claim 1 as set forth above, and that Eleveld et al., 2021 and Seki and Rutz, 2018 both disclose that it is within the realm of routine experimentation for someone of ordinary skill in the art to manipulate the administered ratio of guide RNAs to Cas9 (which is known to be included in vectors expressing only Cas9 and comprising drug-resistance markers, thereby reading on the instantly claimed vector containing a drug-resistance gene as recited at instant claim 1), specifically with ratios of 6:1 gRNA:Cas9 in Eleveld et al., 2021, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to empirically determine the optimal ratio of gRNA vectors to Cas9 vectors (comprising a drug-resistance gene, as instantly claimed) by routine experimentation to predictably optimize the ratio of gRNA vectors to Cas9 vectors, thereby optimizing knockout efficiency (as disclosed in Seki and Rutz, 2018). One would have been motivated to make such a modification in order to receive the expected benefit of optimizing knockout efficiency.
Conclusion
No claims are allowed.
Claims 1, 4, and 10 are objected to.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Sarah E Allen whose telephone number is (571)272-0408. The examiner can normally be reached M-Th 8-5, F 8-12.
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/SARAH E ALLEN/ Examiner, Art Unit 1637
/J. E. ANGELL, Ph.D./ Primary Examiner, Art Unit 1637
Prosecution Insights