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
Applicant’s election without traverse of "a mutated form of the gene encoding neutrophil elastase" as the single mutated gene, and "SEQ ID NO: 1 and SEQ ID NO: 2" as the single first and second sgRNA in the reply filed on May 04, 2026 is acknowledged.
Claims 1-9, and 11-19 are pending and under examination.
Priority
Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55. Acknowledgment is made of applicant’s claim for foreign priority under 35 U.S.C. 119 (a)-(d) based on EPO 21158366.1, which claims filling date of February 22, 2021.
Claim Objections
Claims 6, 9, and 13-14 are objected to because of the following informalities:
In claim 6, line 2, the recitation of “(mutated gene)” proceeds “a mutated form of a wild type gene” as though it is an abbreviation or an acronym. However, “mutated gene” is not a correct abbreviation or acronym for the preceding phrase. If Applicant intends to subsequently refer to a mutated gene, as evidenced by claim 7, it is recommended that claim 6 be amended to “wherein said gene is a mutated gene” so that consistent language is used throughout all the claims.
In claims 9 and 14, line 11, the acronym “sgRNA” is defined as being “single guide RNA”. However, the acronym has previously been defined in claim 1. An acronym should only be defined the first time it appears in an independent claim or in the group of claims under an independent claim.
In claim 13, line 3, the acronym “ELANE” is defined as being “neutrophil elastase”. However, the acronym has previously been defined in claim 7. An acronym should only be defined the first time it appears in an independent claim or in the group of claims under an independent claim.
Appropriate correction is required.
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 1-9, and 11-19 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claims 1 and 9 recites the limitation "the targeted knockout" in line 1. There is insufficient antecedent basis for this limitation in the claim. There is no prior recitation of “targeted knockout”. Thus, it is unclear which element is being further defined in the instant claims.
Claims 17 and 19 recite the limitation “the target nucleic acid” in line 2. There is insufficient antecedent basis for this limitation in the claim. There is no prior recitation of “target nucleic acid”. Thus, it is unclear which element is being further defined in the instant claims.
Those claims included in the statement of rejection but not otherwise discussed are rejected for depending from a rejected claim but failing to remedy the indefiniteness therein.
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claims 17 and 19 are 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. Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Claims 17 and 19 recite “wherein the first and second CRISPR endonucleases do not create double-strand DNA breaks”, and claims 1 and 9 from which claims 17 and 19 depend already recites “said first and said second CRISPR endonucleases are configured to create single-strand DNA breaks”. Nishimasu et al (Crystal structure of Cas9 in complex with guide RNA and target DNA; Cell, 2014, 156: 935-949) teach the CRISPR-associated endonuclease Cas9 contains the HNH and RuvC nuclease domains, which are essential for cleavage of the complementary and noncomplementary strands of the target DNA, respectively (pg. 935, col. 1, para. 1). Thus, CRISPR endonucleases are naturally configured to create double-strand DNA breaks. Further, Nishimasu et al teach alanine substitution of Glu762, His983, Asp986, or D10 in the RuvC domain converts Cas9 into a nickase that cleaves a single strand of DNA by abolishing the cleavage activity of the noncomplementary strand while retaining the cleavage activity of the complementary strand from the HNH nuclease domain (pg. 939, col. 1, para. 3). In other words, specific Cas9 variants or Cas9 nickase that create single-strand DNA breaks inherently do not create double-strand breaks because the double-strand cleavage functionality is abolished in order to become a Cas9 nickase. Claims 17 and 19 recite inherent properties of the CRISPR endonucleases of claims 1 and 9. Thus, claims 17 and 19 fail to further limit the subject matter of the claim from which they depend.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-9, 11-14, 16, and 18 are rejected under 35 U.S.C. 103 as being unpatentable over Ran et al (Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity; Cell, 2013, 154 (6): 1380-1389; IDS received on 08/22/2023, Cite No. 12) in view of Bauer et al (WO2020112979-A2; Published Date: June 04, 2020; IDS received on 09/13/2023, Cite No. 1), and NC_000019.10 (Homo sapiens chromosome 19, GRCh38.p13 Primary Assembly, region: 852190 to 856243; NCBI Reference Sequence; Constructed sequence date: Dec 09, 2019).
For compact prosecution, claims are addressed according to their statutory class. For example, claims drawn to a composition or a method are discussed together, rather than in numerical order.
Regarding claims 1, Ran et al teach a gene editing strategy that combines two CRISPR associated protein 9 (Cas9) nickase (i.e., variant of Cas9) with paired guide RNAs (i.e., first and second guide RNAs) complementary to opposite strands of a genomic DNA sequence (i.e., target nucleic acid) to introduced targeted single-stranded breaks on both sense and antisense strands of the genomic DNA sequence (a portion of Fig. 2A is reproduced below). Thus, Ran et al teach the composition recited in claim 1 to knockout a gene on a double-stranded DNA in a biological cell, comprising (i) a first CRISPR endonuclease and a first sgRNA (e.g., sgRNA a), wherein the first sgRNA is configured to hybridize to the sense strand of the gene; and (ii) a second CRISPR endonuclease and a second sgRNA (e.g., sgRNA b), wherein the second sgRNA is configured to hybridize to the antisense strand of the gene. Ran et al further teach the Cas9 nickase comprises a D10A mutation in the RuvC domain, and individually nick each strand of the DNA to trigger repairs by the high-fidelity base excision repair pathway and minimize off-target activity (pg. 1381, col. 1, para. 2; pg. 1382, col. 1, para. 1). Ran et al further teach this gene editing strategy reduced off-target activity by 50- to 1,500-fold in cell lines when implementing paired sgRNAs spaced by -4 to +23 base pair offset, which is the distance between the two sgRNAs (pg. 1384, col. 1, para. 2-3; pg. 1382, col. 2, para. 1; Figure 2B, left; Figure 3A; Figure 3E).
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However, Ran et al teach targeting various genome locus, including EMX1, DYRK1A, and VEGFA (Figure 5B and Figure 3E-F). Ran et al do not teach targeting a mutated gene encoding neutrophil elastase (ELANE) and wherein the first or second sgRNA comprises the nucleotide sequence of elected SEQ ID NOs: 1 and 2.
Bauer et al teach a composition that comprises (i) a CRISPR Cas9 endonuclease and (ii) guide RNAs complementary to the promoter driving expression of the ELANE gene ([0019], Table 1). Bauer et al further teach using this composition for “ex vivo targeted genome editing of the ELANE gene in a progenitor cell” to decrease ELANE mRNA or protein expression ([0016], [0027]), demonstrated by therapeutic gene editing of a mutated form of the gene encoding ELANE in human hematopoietic stem cells that originated from a subject with the mutated ELANE gene ([0106], [0109], FIG. 31, FIG. 34; FIG. 3 to 5). Particularly, Bauer et al teach two sgRNAs, SEQ ID NO: 178 that is 100% identical to instantly claimed SEQ ID NO: 1, and SEQ ID NO: 81 that is 100% identical to instantly claimed SEQ ID NO: 2 (see sequence alignment below) ([0019], Table 1). Supported by NC_000019.10, the first sgRNA SEQ ID NO: 178 is complementary to the sense strand (e.g., ++) at region starting from 852,268 to 852,287 while the second sgRNA SEQ ID NO: 81 is complementary to the antisense strand (e.g., -+) at region starting from 852,240 to 852,259, which both hybridizes to the promoter upstream of the ELANE gene ranging from 852,303 to 856,243 (i.e., hybridizing to a genetic element controlling the expression of said gene) (see sequence alignment below). The distance or offset between the hybridization site of the two sgRNA are 9 bases (852,268-852,259 = 9), which falls within the optimal offset range of -4 to +23 base pairs set in the gene editing strategy of Ran et al. Further, Bauer et al demonstrate Cas9 and sgRNAs SEQ ID NO: 178 and SEQ ID NO: 81 targeting mutated ELANE gene successfully yielded neutrophil maturation and increased promyelocytes (FIG. 11)
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Thus, it would have been obvious to one of ordinary skill in the art before the effective filling date of the invention to have modified the paired sgRNAs of Ran et al with sgRNAs targeting the promoter of a mutated form of the gene encoding ELANE as taught by Bauer et al because it would have merely amounted to a simple substitution of prior art elements according to known methods to yield predictable results. The substituted component (sgRNAs targeting EMX1 locus to sgRNAs targeting the promoter of ELANE) and its functions (localizing the coupled Cas9 to the desired target site) were known in the art. The particular sgRNA pair would have been an obvious selection because Bauer et al identifies the same target region of the ELANE promoter, and publicly available genomic sequence information NC_000019.10 demonstrates the recited sgRNA pair of Bauer et al are complementary to opposite DNA strands with an approximately 8 base pair spacing, which falls within the optimal sgRNA offset spacing taught by Ran et al for efficient and specific nickase cleavage. One would have been motivated to have done so for the advantage of targeting the promoter region of a mutated ELANE gene that is a desirable therapeutic target for treatment of ELANE-associated diseases as taught by Bauer et al ([0003]-[0008], Table 1), and to apply the gene editing strategy of Ran et al to therapeutically relevant genomic locus while obtaining the reduced off-target activity taught by Ran et al. One would have had a reasonable expectation of success in doing so because Rant et al teach paired sgRNAs and CRISPR Cas9 nickase function by creating single-strand DNA breaks at opposite strands on the desired genomic locus, while Bauer et al establishes that the promoter of a mutated ELANE gene is an effective CRISPR target site via the recited sgRNA pairs. Thus, substituting the sgRNA pair of Ran et al with the specific sgRNAs of Bauer et al would have been a predictable use of a known CRISPR gene editing strategy according to its established functions.
Regarding claim 2, the obviousness to modify the first and second guide RNAs of Ran et al to guide RNAs taught by Bauer et al is discussed above as applied to claim 1. Bauer et al in view of NC_000019.10 teach the first and second guide RNAa are configured to hybridize to genetic element controlling the expression the ELANE gene is a promoter, see sequence alignment to NC_000019.10 provided above as applied to claim 1.
Regarding claims 3-5, Ran et teach the CRISPR endonuclease is a Cas9 D10A nickase (pg. 1381, col. 1, para. 2; pg. 1382, col. 1, para. 1) as discussed above as applied to claim 1.
Regarding claims 6 and 7, the obviousness to modify the first and second guide RNAs of Ran et al to guide RNAs taught by Bauer et al is discussed above as applied to claim 1. Bauer et al teach that first and second guide RNAs are configured to hybridize to a mutated form of a gene encoding ELANE ([0106], [0109], [0019], Table 1) as discussed above as applied to claim 1.
Regarding claim 8, the obviousness to modify the first and second guide RNAs of Ran et al to guide RNAs taught by Bauer et al is discussed above as applied to claim 1. Bauer et al teach wherein the guide RNAs comprise of SEQ ID NO: 178 and SEQ ID NO: 81, which are 100% identical to instant SEQ ID NO: 1 and SEQ ID NO: 2, respectively (see sequence alignment provided above in claim 1).
Regarding claim 16, Ran et al teach the CRISPR nickase are encoded in plasmid (i.e., DNA) for transfection into human embryonic stem cell lines (pg. 1387, col. 2, subsection “Cell Culture and Transfection”).
Regarding claims 9, the obviousness to modify the sgRNA pairs of Ran et al with instantly claimed sgRNAs comprising the sequence of SEQ ID NO: 1 and SEQ ID NO: 2 is discussed above and as applied to claim 1. Ran et al further teach a method for targeted knockout of a gene on a double-stranded DNA in a biological cell, comprising the following steps:
1) providing a biological cell comprising a gene on double-stranded DNA (e.g., human embryonic kidney cell line or human embryonic stem cell line) (pg. 1387, col. 2, subsection “Cell Culture and Transfection”);
2) introducing into said biological cell the composition of claim 1 (e.g., cells were transfected with plasmids (i.e., nucleic acid molecules, DNA) encoding the CRISPR Cas9 nickase and sgRNA pairs) (pg. 1387, col. 2, subsection “Cell Culture and Transfection”);
3) incubating said cell and said composition (e.g., cells were incubated at 37°C for 72 hours post transfection prior to genomic DNA extraction to validate DNA cleavage efficiency (pg. 1387, col. 2, subsection “SURVEYOR Nuclease Assay for Genome Modification”).
Similarly, Bauer et al teach a method for targeted knockout of a gene on a double-stranded DNA in a biological cell comprising the same steps: 1) providing an isolated hematopoietic progenitor cell or a hematopoietic stem cell (claim 34); 2) introducing nucleic acid molecules encoding CRISPR Cas9 nickase and sgRNA pairs (claim 32); and 3) resuspending electroporated cells in media for at least 24 hours ([0255]).
Regarding claim 11, the teachings of Bauer et al are discussed above as applied to claim 9. Bauer et al further teach that the biological cell is a hematopoietic stem and progenitor cell (claim 34).
Regarding claims 12 and 13, Bauer et al further teach the biological cell originates from a subject with a mutated gene encoding ELANE ([0106], [0109], FIG. 31, FIG. 34; FIG. 3 to 5) as discussed above as applied to claims 1, 6, and 7.
Regarding claim 18, Ran et al teach that nucleic acid molecule encoding the CRISPR endonuclease is DNA (pg. 1387, col. 2, subsection “Cell Culture and Transfection”) as discussed above as applied to claim 9.
Regarding claim 14, the teachings of a method for targeted knockout of a gene on double-stranded DNA in a biological cell are discussed above and as applied to claim 9. Ran et al further teach cells are incubated at 37°C for 72 hours after transfection, following genomic DNA extraction, which under the reasonable interpretation, the cells are recovered prior to performing DNA extraction (pg. 1387, col. 2, subsection “SURVEYOR Nuclease Assay for Genome Modification”). In addition, Ran et al also teach transcribing Cas9 nickase and sgRNAs, microinjecting the Cas9 nickase mRNAs and sgRNAs into mouse zygotes (i.e., steps 1 and 2), incubating the mouse zygotes for 6 days (i.e., step 3), and individually collecting the now expanded blastocysts for downstream genome extraction (i.e., step 4) (pg. 1388, subsections “Microinjection into Mouse Zygotes” and “Genome Extraction from Blastocyst Embryos”). In addition, Bauer et al also teach recovering the electroporated cells for in vitro differentiation ([0257]).
Claim 15 is rejected under 35 U.S.C. 103 as being unpatentable over Ran et al (Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity; Cell, 2013, 154 (6): 1380-1389; IDS received on 08/22/2023, Cite No. 12) in view of Bauer et al (WO2020112979-A2; Published Date: June 04, 2020; IDS received on 09/13/2023, Cite No. 1), and NC_000019.10 (Homo sapiens chromosome 19, GRCh38.p13 Primary Assembly, region: 852190 to 856243; NCBI Reference Sequence; Constructed sequence date: Dec 09, 2019) as applied to claim 1, and further in view of Zhang (US 8,697,359 B1; Published Date: Apr 15, 2021)
Regarding claim 15, the obviousness to modify the sgRNA pairs of Ran et al with instantly claimed sgRNAs comprising the sequence of SEQ ID NO: 1 and SEQ ID NO: 2 is discussed above and as applied to claim 1. The teachings of Ran et al regarding instructions to deliver the composition to a biological cell are discussed above and as applied to claims 9 and 14.
However, Ran et al do not teach a kit comprising the composition of claim 1.
Zhang teaches a method of altering expression of target gene sequences and related gene products using a composition comprising CRISPR Cas9 nuclease and sgRNAs (col. 2, para. 3; col. 3, para. 2). Zhang further teach this composition or a vector system comprising nucleic acids encoding each component of this composition is comprised in a kit, along with instructions for using the kit (col. 6, para. 2).
Thus, it would have been obvious to one of ordinary skill in the art before the effective filling date of the invention to have modified the composition of Ran et al to be in a kit because it would have merely amounted to a simple combination of prior art elements according to known methods to yield predictable results. Each component in the combination performs the same function as they do separately. The combination of individual elements in a kit does not change function of each element separately, and a kit is merely a collection of elements for a given method. One would have been motivated to have done so for the advantage of allowing users to practice the method in a more convenient way. One would have had a reasonable expectation of success in doing so because a kit merely represents a predictable variation and Zhang teaches facilitating components of the composition in a kit to practice the gene editing method.
Conclusion
No claims are allowable.
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/QIWEN SU-TOBON/
Examiner
Art Unit 1636
/NEIL P HAMMELL/Supervisory Patent Examiner, Art Unit 1636
Prosecution Insights