DETAILED ACTION
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 with traverse of Group II (claims 4-15) in the reply filed on 02/18/2026 is acknowledged. The traversal is on the ground(s) that Groups I and II are unified due to sharing a single inventive concept of improving prime-editing efficiency/target specificity by enhancing chromatin accessibility using proximal dsgRNA and/or chromatin-modulating peptides. This is not found persuasive because all the components of genome editing compositions comprising a fusion protein including a CRISPR/Cas9 protein, a reverse transcriptase, a pegRNA, and dsgRNAs were disclosed in the art prior to the effective filing date of the instant application. As set forth in the restriction requirement dated 12/18/2025, it would have been obvious to someone of ordinary skill in the art prior to the effective filing date of the claimed invention to include the dsgRNAs disclosed in Liu et al., 2019 (as cited in Applicant IDS; of record) in the prime editing composition of Anzalone et al., 2019 (as cited in Applicant IDS; of record) to predictably increase the editing efficiency of the Cas9 fusion protein disclosed in Anzalone et al., 2019. Furthermore, the gene editing composition of Group II requires chromatin-modulating peptides that are not required by Group I. Accordingly, Groups I-II are not so linked by the same or a corresponding special technical feature as to form a single general inventive concept.
The requirement is still deemed proper and is therefore made FINAL.
Claims 1-3, 16, and 17 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected invention, there being no allowable generic or linking claim. Applicant timely traversed the restriction (election) requirement in the reply filed on 02/18/2026.
Accordingly, claims 4-15 are pending and under consideration.
Priority
Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55. The earliest effective filing date to which the instant application is entitled is 09/24/2020.
Information Disclosure Statement
Receipt of information disclosure statements on 03/23/2023 and 03/06/2025 is acknowledged. The signed and initialed PTO-1449‘s have been mailed with this action.
Drawings
The drawings filed 03/23/2023 are acceptable.
Nucleotide and/or Amino Acid Sequence Disclosures
REQUIREMENTS FOR PATENT APPLICATIONS CONTAINING NUCLEOTIDE AND/OR AMINO ACID SEQUENCE DISCLOSURES
Items 1) and 2) provide general guidance related to requirements for sequence disclosures.
37 CFR 1.821(c) requires that patent applications which contain disclosures of nucleotide and/or amino acid sequences that fall within the definitions of 37 CFR 1.821(a) must contain a "Sequence Listing," as a separate part of the disclosure, which presents the nucleotide and/or amino acid sequences and associated information using the symbols and format in accordance with the requirements of 37 CFR 1.821 - 1.825. This "Sequence Listing" part of the disclosure may be submitted:
In accordance with 37 CFR 1.821(c)(1) via the USPTO patent electronic filing system (see Section I.1 of the Legal Framework for Patent Electronic System (https://www.uspto.gov/PatentLegalFramework), hereinafter "Legal Framework") as an ASCII text file, together with an incorporation-by-reference of the material in the ASCII text file in a separate paragraph of the specification as required by 37 CFR 1.823(b)(1) identifying:
the name of the ASCII text file;
ii) the date of creation; and
iii) the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(1) on read-only optical disc(s) as permitted by 37 CFR 1.52(e)(1)(ii), labeled according to 37 CFR 1.52(e)(5), with an incorporation-by-reference of the material in the ASCII text file according to 37 CFR 1.52(e)(8) and 37 CFR 1.823(b)(1) in a separate paragraph of the specification identifying:
the name of the ASCII text file;
the date of creation; and
the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(2) via the USPTO patent electronic filing system as a PDF file (not recommended); or
In accordance with 37 CFR 1.821(c)(3) on physical sheets of paper (not recommended).
When a “Sequence Listing” has been submitted as a PDF file as in 1(c) above (37 CFR 1.821(c)(2)) or on physical sheets of paper as in 1(d) above (37 CFR 1.821(c)(3)), 37 CFR 1.821(e)(1) requires a computer readable form (CRF) of the “Sequence Listing” in accordance with the requirements of 37 CFR 1.824.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed via the USPTO patent electronic filing system as a PDF, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the PDF copy and the CRF copy (the ASCII text file copy) are identical.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed on paper or read-only optical disc, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the paper or read-only optical disc copy and the CRF are identical.
Specific deficiencies and the required response to this Office Action are as follows:
►Specific deficiency – Nucleotide and/or amino acid sequences appearing in the drawings are not identified by sequence identifiers in accordance with 37 CFR 1.821(d). Sequence identifiers for nucleotide and/or amino acid sequences must appear either in the drawings or in the Brief Description of the Drawings. See Figures 1A, 2A, 4B, 4C, 8A, 8B, 8C and 9B.
Required response – Applicant must provide:
Replacement and annotated drawings in accordance with 37 CFR 1.121(d) inserting the required sequence identifiers;
AND/OR
A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3) and 1.125 inserting the required sequence identifiers into the Brief Description of the Drawings, consisting of:
A copy of the previously-submitted specification, with deletions shown with strikethrough or brackets and insertions shown with underlining (marked-up version);
A copy of the amended specification without markings (clean version); and
A statement that the substitute specification contains no new matter.
Specification
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code. See paragraph [0123]. Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01.
The lengthy specification has not been checked to the extent necessary to determine the presence of all possible minor errors. Applicant’s cooperation is requested in correcting any errors of which applicant may become aware in the specification.
Claim Objections
Claims 6, 8, and 15 are objected to because of the following informalities:
Claim 6 recites that “the chromatin-modulating peptide is linked to the CRISPR/Cas9 protein or the reverse transcriptase directly by a chemical bond, indirectly by a linker, or in combination thereof” (bolded emphasis added). The phrase “in combination thereof” is awkward and not standard. For purposes of ease of reading the instant claim set, it would be remedial to recite a more standard phrase, such as “a combination thereof” (bolded emphasis added). This is merely an example set forth by the Examiner that is not intended to be limiting.
With regard to claim 8, which recites “a N-terminus” (bolded emphasis added), the article “a” is grammatically improper when preceding words articulated with an initial vowel sound. While “N-terminus” is spelled such that it recites a consonant letter “N,” the pronunciation of the “N” in “N-terminus” nonetheless begins with a vowel sound. It would be remedial to update the instant claim language to be grammatically proper.
Claim 15 recites “the gene editing composition of claim 4, further comprising: single guide RNA (sgRNA)…”, which does not comport with standard grammatical and/or linguistic conventions. The recitation of a single guide RNA (sgRNA) is not preceded by an article such as “a,” as is conventional. It would be remedial to amend the instant claim to recite “the gene editing composition of claim 4, further comprising: a single guide RNA (sgRNA)…” (bolded and underlined emphasis added) such that it comports with standard grammatical and/or linguistic conventions. This is merely an example set forth by the Examiner that is not intended to be limiting.
Appropriate correction is required.
Claim Rejections - 35 USC § 112(a)
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 4-8 and 11-15 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.
Claim 4 (from which all other claims directly or indirectly depend) is drawn to a set of gene editing compositions comprising a fusion protein and guide RNAs, wherein said fusion protein comprises a CRISPR/Cas9 protein or a variant thereof and a reverse transcriptase or a variant thereof, and wherein the guide RNAs include a prime editing guide RNA (pegRNA) and a dead single guide RNA (dsgRNA). The rejected claims thus comprise a set of gene editing compositions that encompass any CRISPR/Cas9 protein or variant thereof, wherein said CRISPR/Cas9 protein or variant thereof must be capable of functioning with a pegRNA and a dsgRNA. Additionally, dependent claim 15 requires that the CRISPR/Cas9 protein or variant thereof must induce cleavage of a target DNA strand upon binding of an sgRNA that complementarily binds to a non-target DNA strand.
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 experimentation with a gene editing composition comprising a nickase variant of Cas9 (nCas9) (paragraphs [0109], [0133], and [0141]). No description is provided of experimentation with any other CRISPR/Cas9 protein or variant thereof.
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 nickase Cas9. The results are not necessarily predictive of any other CRISPR/Cas9 protein or variant thereof, including nuclease active Cas9. Thus, it is impossible for one to extrapolate from the single nCas9 species described herein those CRISPR/Cas9 proteins or variants thereof that would necessarily meet the structural/functional characteristics of the rejected claims.
The prior art does not appear to offset the deficiencies of the instant specification. As is known to those of ordinary skill in the art, Cas9 comprises two nuclease domains: RuvC and HNH (reviewed in Jo et al., 2015: see Figure 1). These two nuclease domains mediate double-stranded cleavage upon target recognition by a single guide RNA (sgRNA). The RuvC and HNH nuclease domains may be separately inactivated (i.e. D10A inactivates RuvC and H840A inactivates HNH) to generate nickase Cas9 variants (reviewed in Jo et al., 2015: see Figure 2). As depicted in Figure 2 of Jo et al., 2015, D10A Cas9 nickase mutants cleave the strand targeted by the sgRNA, while H840A Cas9 nickase mutants cleave the strand complementary to the strand targeted by the sgRNA. Further, as is known to those of ordinary skill in the art, pegRNAs are a crucial component of prime editing, along with prime editors, which comprise a Cas9 nickase domain fused to a reverse transcriptase domain (reviewed in Anzalone et al., 2019; cited in Applicant IDS; of record; see Figure 1). This nickase function is crucial to the function of pegRNAs, which prime reverse transcription of new DNA containing the desired edit using the reverse transcriptase template within said pegRNA (reviewed in Anzalone et al., 2019; cited in Applicant IDS; of record; see Figure 1). In comparison, nuclease active Cas9 mediates double-strand breaks, which may be resolved by homology directed repair or by non-homologous end joining, thereby resulting in an insertion, deletion, or mutation of specific DNA sequences (reviewed in Jo et al., 2015: see Figure 1 and page 235, column 1, paragraph 2). This is an entirely distinct process from the prime editing accomplished via prime editors (comprising a Cas9 nickase domain fused to a reverse transcriptase domain) guided by pegRNAs (reviewed in Anzalone et al., 2019; cited in Applicant IDS; of record; see Figure 1).
Therefore, the skilled artisan would have reasonably concluded applicants were not in possession of the claimed invention for claims 4-8 and 11-15. Dependent claims 9 and 10 require that the CRISPR/Cas9 protein or variant thereof is a nickase variant, which is compatible with pegRNAs, as set forth above. Accordingly, dependent claims 9 and 10 do not inherit the rejection of independent claim 4.
Claim Rejections - 35 USC § 112(b)
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 4-6 and 8-15 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.
With regard to claim 4, from which all other claims directly or indirectly depend, under broadest reasonable interpretation, the recitation of “chromatin-modulating peptides” (bolded and underlined emphasis added) encompasses multiple chromatin-modulating peptides. However, dependent claims 5 and 6 respectively further recite that “the chromatin-modulating peptide is a high-mobility group nucleosome binding domain 1 (HN1), a histone H1 central globular domain (H1G), or a combination thereof” (bolded and underlined emphasis added) and that “the chromatin-modulating peptide is linked to the CRISPR/Cas9 protein” (bolded and underlined emphasis added). Thus, the instant claim set encompasses two, mutually exclusive interpretations under broadest reasonable interpretation: one in which the gene editing composition requires at least two chromatin-modulating peptides, and one in which the gene editing composition requires at least one chromatin-modulating peptide. Dependent claims 8-15 do not further clarify which interpretation is being claimed, and thus they inherit the rejection of claim 4. Dependent claim 7 requires two chromatin-modulating peptides and is therefore not included in this rejection.
In order to ensure that one of ordinary skill in the art is reasonably apprised of the bounds of protection sought by the instant claim set, it would be remedial to amend independent claim 4 to clearly recite whether the gene editing composition requires at least two chromatin-modulating peptides or whether the gene editing composition requires at least one chromatin-modulating peptide.
For purposes of examination and in the interest of compact prosecution, the Examiner has interpreted instant claim 4 to require at least one chromatin-modulating peptide, as recited at claim 5.
With further regard to claim 4, the recitation of “a guide RNA…wherein the guide RNA includes prime editing guide RNA (pegRNA) and dead single guide RNA (dsgRNA)” (bolded and underlined emphasis added), under broadest reasonable interpretation, the instant claim encompasses two, mutually exclusive interpretations: one in which the gene editing composition requires one guide RNA (i.e. “a guide RNA”), and one in which the gene editing composition requires two species of guide RNA (i.e. a pegRNA and a dsgRNA).
In order to ensure that one of ordinary skill in the art is reasonably apprised of the bounds of protection sought by the instant claim set, it would be remedial to amend independent claim 4 to clearly recite whether the gene editing composition requires one guide RNA or whether the gene editing composition requires two guide RNAs: one pegRNA and one dsgRNA.
For purposes of examination and in the interest of compact prosecution, the Examiner has interpreted instant claim 4 to require two guide RNAs: one pegRNA and one dsgRNA, as is embraced by the instant claim language.
With specific regard to claim 8, the recitation of “the fusion protein further includes a nuclear localization signal at a N-terminus and C-terminus, respectively” (bolded and underlined emphasis added) is indefinite, as under broadest reasonable interpretation, the instant claim embraces the inclusion of one and two nuclear localization signals. If a nuclear localization signal is fused to both the N-terminus and C-terminus there must necessarily be two nuclear localization signals. However, the instant claim recites a single nuclear localization signal.
In order to ensure that one of ordinary skill in the art is reasonably apprised of the bounds of protection sought by the instant claim set, it would be remedial to amend the instant claim to clearly recite whether the gene editing composition requires a single nuclear localization signal at either the N-terminus or the C-terminus, or whether the gene editing composition requires two nuclear localization signals at both the N-terminus and the C-terminus.
For purposes of examination and in the interest of compact prosecution, the Examiner has interpreted instant claim 8 to require a single nuclear localization signal at either the N-terminus or the C-terminus, as the instant claim recites “a nuclear localization signal” (bolded and underlined emphasis added).
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.
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 4-11 and 13-15 are rejected under 35 U.S.C. 103 as being unpatentable over US 2023/0242899 A1 (hereinafter Steinberg; effectively filed 06/05/2020) in view of US 2019/0249200 A1 (hereinafter Seebeck) and Liu et al., 2019 (hereinafter Liu; as cited in Applicant IDS; of record), as evidenced by Jo et al., 2015 (hereinafter Jo).
With regard to claim 4, which recites “a gene editing composition comprising:
a fusion protein or a nucleic acid encoding the fusion protein, including i) a CRISPR/Cas9 protein or a variant thereof, ii) a reverse transcriptase or a variant thereof, and iii) chromatin-modulating peptides; and
a guide RNA or a nucleic acid encoding the guide RNA, wherein the guide RNA includes prime editing guide RNA (pegRNA) and dead single guide RNA (dsgRNA),”
Steinberg discloses compositions for altering genomes (paragraph [0004]), said compositions comprising fusion of a CRISPR/Cas9 protein or a variant thereof to a reverse transcriptase domain (Figures 29A and 29B), wherein said CRISPR/Cas9 protein or a variant thereof is guided to a target locus by a pegRNA (paragraph [0228]). Steinberg discloses that the system taught therein facilitates introduction of exogenous genetic elements into a targeted locus of a host genome (paragraph [0004]). However, Steinberg is silent as to the instantly claimed chromatin-modulating peptide and dsgRNA.
These deficiencies are cured by Seebeck and Liu, as set forth below.
Seebeck discloses engineered Cas9 systems for precision editing, said Cas9 systems comprising Cas9 fusion to one or more heterologous domains, including chromatin modulating motifs, such as nucleosome interacting peptides derived from high mobility group proteins (i.e. HMGN1) or the central globular domain of histone variants (i.e. H1G) (abstract; paragraphs [0005], [0025], and [0029]). Seebeck further discloses that CMM fusion enhances Cas9 activity (paragraph [0107]).
Liu discloses that dsgRNAs improve editing efficiency of Cas9 and fusion proteins thereof (such as the Cas9-TV fusion protein taught therein) up to several folds (abstract; page 2, column 2, paragraph 2; page 4, column 1-section “Proximal targeting with dsgRNA improves genome editing by Cas9-TV and Cas9”).
Thus, it is considered that Steinberg, Seebeck, and Liu collectively disclose each and every limitation of instant claim 4.
With regard to claim 5, which recites “the chromatin-modulating peptide [of the gene editing composition of claim 4] is a high-mobility group nucleosome binding domain 1 (HN1), a histone H1 central globular domain (H1G), or a combination thereof,” as set forth above, Seebeck discloses engineered Cas9 systems for precision editing, said Cas9 systems comprising Cas9 fusion to one or more heterologous domains, including chromatin modulating motifs, such as nucleosome interacting peptides derived from high mobility group proteins (i.e. HMGN1) or the central globular domain of histone variants (i.e. H1G) (abstract; paragraphs [0005], [0025], and [0029]). Thus, Seebeck discloses each and every additional limitation of instant claim 5.
With regard to claim 6, which recites “the chromatin-modulating peptide [of the gene editing composition of claim 4] is linked to the CRISPR/Cas9 protein or the reverse transcriptase directly by a chemical bond, indirectly by a linker, or in combination thereof,” as set forth above, Seebeck discloses engineered Cas9 systems for precision editing, said Cas9 systems comprising Cas9 fusion to one or more heterologous domains, including chromatin modulating motifs, such as nucleosome interacting peptides derived from high mobility group proteins (i.e. HMGN1) or the central globular domain of histone variants (i.e. H1G) (abstract; paragraphs [0005], [0025], and [0029]). Seebeck further discloses that the one or more heterologous domains taught therein may be fused to the Cas9 taught therein directly by a chemical bond or indirectly via linkers (paragraph [0025]). Thus, Seebeck discloses each and every additional limitation of instant claim 6.
With regard to claim 7, which recites “the fusion protein [of the gene editing composition of claim 4] consists of N-terminus-[HN1]-[Cas9]-[H1G]-reverse transcriptase]-C-terminus; or N-terminus-[HN1]-[Cas9]-[reverse transcriptase]-[H1G]-C-terminus,” as set forth above, Steinberg, Seebeck, and Liu collectively disclose the fusion protein of instant claim 4. While the references do not disclose the particular order in which the components of the fusion protein are assembled, per MPEP § 2144.04(VI)(C), it is not inventive to rearrange parts when those parts were all disclosed in the prior art. Thus, it is considered that Steinberg, Seebeck, and Liu collectively disclose each and every limitation of instant claim 7.
With regard to claim 8, which recites “the fusion protein [of the gene editing composition of claim 4] further includes a nuclear localization signal at a[n] N-terminus and C-terminus, respectively,” Seebeck further discloses that the Cas9 fusions taught therein may comprise nuclear localization signals at either the N-terminus, the C-terminus, or any combination thereof (paragraphs [0025] and [0026]). Thus, Seebeck discloses each and every additional limitation of instant claim 8.
With regard to claim 9, which recites “the CRISPR/Cas9 protein variant [of the gene editing composition of claim 4] is a nickase,” Seebeck further discloses that the Cas9 of the fusion proteins taught therein may be engineered such that one or both of the RuvC and HNH domains is/are inactivated to generate a nickase Cas9 or a dead Cas9 (paragraph [0023]). Thus, Seebeck discloses each and every additional limitation of instant claim 9.
With regard to claim 10, which recites “in the CRISPR/Cas9 protein variant [of the gene editing composition of claim 9], either a RuvC domain or an HNH domain is deactivated, as set forth above, Seebeck further discloses that the Cas9 of the fusion proteins taught therein may be engineered such that one or both of the RuvC and HNH domains is/are inactivated to generate a nickase Cas9 or a dead Cas9 (paragraph [0023]). Thus, Seebeck discloses each and every additional limitation of instant claim 10.
With regard to claim 11, which recites “the reverse transcriptase or the variant thereof [of the gene editing composition of claim 4] is derived from a Moloney murine leukemia virus (M-MLV),” as set forth above, Steinberg discloses compositions for altering genomes (paragraph [0004]), said compositions comprising fusion of a CRISPR/Cas9 protein or a variant thereof to a reverse transcriptase domain (Figures 29A and 29B), wherein said CRISPR/Cas9 protein or a variant thereof is guided to a target locus by a pegRNA (paragraph [0228]). Steinberg further discloses that the reverse transcriptase domain taught therein may be derived from MMLV (paragraph [0153]). Thus, Steinberg discloses each and every additional limitation of instant claim 11.
With regard to claim 13, which recites “the dsgRNA [of the gene editing composition of claim 4] consists of 10 to 20 nucleotides (nt) in length,” as set forth above, Liu discloses that dsgRNAs improve editing efficiency of Cas9 and fusion proteins thereof (such as the Cas9-TV fusion protein taught therein) up to several folds (abstract; page 2, column 2, paragraph 2; page 4, column 1-section “Proximal targeting with dsgRNA improves genome editing by Cas9-TV and Cas9”). Liu further discloses that the dsgRNAs utilized therein are 14 to 15 nucleotides in length (page 4, column 1, paragraph 2). Given that the narrow range of 14 to 15 nucleotides falls within the broader instantly claimed range of 10 to 20 nucleotides, Liu discloses each and every additional limitation of instant claim 13.
With regard to claim 14, which recites “the dsgRNA [of the gene editing composition of claim 4] binds to a location of 5 to 70 nt away from a pegRNA binding site to increase chromatin accessibility of the fusion protein,” as set forth above, Liu discloses that dsgRNAs improve editing efficiency of Cas9 and fusion proteins thereof (such as the Cas9-TV fusion protein taught therein) up to several folds (abstract; page 2, column 2, paragraph 2; page 4, column 1-section “Proximal targeting with dsgRNA improves genome editing by Cas9-TV and Cas9”). Liu further discloses that to optimize proximal dsgRNA targeting, they designed dsgRNAs targeting sites from 47 to 266 bp from the sgRNA target site (Figure 5) and that such proximal dsgRNA targeting increases local chromatin accessibility (Figure 6; section “Cas9-TV together with proximal dsgRNA increases chromatin accessibility). While Liu does not disclose the instantly claimed pegRNA, as set forth above, Steinberg discloses the utility of including pegRNAs in gene editing compositions (paragraph [0228]). Thus, it is considered that Liu and Steinberg collectively disclose each and every additional limitation of instant claim 14.
With regard to claim 15, which recites “the gene editing composition of claim 4, further comprising: single guide RNA (sgRNA) that complementarily binds to a non-target DNA strand to induce cleavage of a target DNA strand,” as set forth above, Liu discloses at figure 5 that to optimize proximal dsgRNA targeting, they designed dsgRNAs targeting sites from 47 to 266 bp from the sgRNA target site. While Liu does not explicitly disclose that the sgRNA taught therein complementarily binds to a non-target DNA strand to induce cleavage of a target DNA strand, those of ordinary skill in the art are aware that sgRNAs facilitate cleavage of both the targeted and non-targeted DNA strands when the Cas9 has nuclease activity, and that H840A Cas9 nickases (encompassed by the instantly claimed CRISPR/Cas9 protein or variant thereof) cleave only the DNA strand that is noncomplementary to the sgRNA (Figure 2). In both embodiments, the sgRNA induces cleavage of a target DNA strand. Thus, it is considered that Liu discloses each and every additional limitation of instant claim 15, as evidenced by Jo.
Given that Steinberg discloses editing compositions comprising a Cas9 fused to a reverse transcriptase (i.e. derived from MMLV) and comprising a pegRNA to simultaneously target the genome and insert sequences of interest, that Seebeck discloses editing compositions comprising a Cas9 (or a nickase thereof) fused to chromatin-modulating peptides, thereby improving Cas9 activity, and that Liu discloses that inclusion of dsgRNAs improve Cas9 editing efficiency, 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 system of Steinberg to include fusion of chromatin-modulating peptides and to include dsgRNA(s) to predictably improve Cas9 activity and efficiency for targeted gene modification/editing. One would have been motivated to make such a modification in order to receive the expected benefit of more effectively and efficiently performing targeted gene modification/editing.
Conclusion
No claims are allowed.
Claims 6, 8, and 15 are objected to.
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/ Primary Examiner, Art Unit 1637
Prosecution Insights