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 Group III (claims 50-65) in the reply filed on April 15, 2026 is acknowledged. Claims 66-68 are new and support is found in claims 52, 53, and 57 filed on August 2, 2024.
Applicant’s election without traverse of SEQ ID NO: 40 as the single specific amino acid sequence for the Cas12i2 domain, SEQ ID NO: 61 as the single amino acid sequence for the fusion domain, and an NLS as the second heterologous sequence in the reply filed on April 15, 2026 is acknowledged.
Claims 53 and 68 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected species, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on April 15, 2026.
Claims 50-52, 54-67 are pending and under examination.
Priority
Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged. Applicant has not complied with one or more conditions for receiving the benefit of an earlier filing date under 35 U.S.C. 119(e) as follows:
The later-filed application must be an application for a patent for an invention which is also disclosed in the prior application (the parent or original nonprovisional application or provisional application). The disclosure of the invention in the parent application and in the later-filed application must be sufficient to comply with the requirements of 35 U.S.C. 112(a) or the first paragraph of pre-AIA 35 U.S.C. 112, except for the best mode requirement. See Transco Products, Inc. v. Performance Contracting, Inc., 38 F.3d 551, 32 USPQ2d 1077 (Fed. Cir. 1994).
The disclosure of the prior-filed application, Application No. 63/139,651, fails to provide adequate support or enablement in the manner provided by 35 U.S.C. 112(a) or pre-AIA 35 U.S.C. 112, first paragraph for one or more claims of this application. This application discloses a Cas12i2 domain comprising an amino acid sequence with at least 95% identity to SEQ ID NO: 40 (pg. 61, lines 25-29; Table 3), but the application does not disclose a heterologous sequence comprising an amino acid sequence with at least 88% identity to SEQ ID NO: 61 (i.e., KRPAATKKAGQAKKKK). The application discloses “may comprise a NLS such as an SV40, c-Myc-NLS, or other suitable monopartite NLS” (pg. 25, lines 9-10). Makkerh et al (Comparative mutagenesis of nuclear localization signals reveals the importance of neutral and acidic amino acids; Current Biology, 1996, 6(8): 1025-1027) teach instant SEQ ID NO: 61 is a nuclear localization signal (NLS) of nucleoplasmin, and it is bipartite requiring two essential clusters of basic amino acids separated by a mutation-tolerant spacer (abstract). Makkerh et al further teach the NLS of simian virus 40 large T-antigen (SV40) requires a single cluster of basic amino acids PKKKRKV (abstract), which positively teaching the of SV40 is different than SEQ ID NO:61. The application further discloses by reference of US2015-0246139, particularly Table 6 (pg. 31, lines 11-12), but an ABSS search alignment of SEQ ID NO: 61 did not show any match results to '139 or its corresponding published US patent 9283287. Therefore, this application fails to provide written description support for a heterologous sequence of SEQ ID NO: 61, and as a result also fails to disclose a Cas12i2 fusion protein of instant claim 1.
The disclosure of the prior-filed application, Application No. 63/227,404, fails to provide adequate support or enablement in the manner provided by 35 U.S.C. 112(a) or pre-AIA 35 U.S.C. 112, first paragraph for one or more claims of this application. This application discloses a Cas12i2 domain comprising an amino acid sequence with at least 95% identity to SEQ ID NO: 40 (pg. 68, lines 19-21; Table 3), but the application does not disclose a heterologous sequence comprising an amino acid sequence with at least 88% identity to SEQ ID NO: 61. The application discloses “may comprise a NLS such as an SV40, c-Myc-NLS, or other suitable monopartite NLS” (pg. 25, lines 15-16), and “in some embodiments the NLS is monopartite or bipartite” (pg. 33, lines 13-14). The application further discloses by reference of US2015-0246139, particularly Table 6 (pg. 33, lines 21-23), but an ABSS search alignment of SEQ ID NO: 61 did not show any match results to '139 or its corresponding published US patent 9283287. Further, the teachings of Makkerh et al regarding SEQ ID NO: 61 is discussed above as applied to application ‘651. Therefore, this application fails to provide written description support for a heterologous sequence of SEQ ID NO: 61, and as a result also fails to disclose a Cas12i2 fusion protein of instant claim 1.
The disclosure of the prior-filed application, Application No. 63/270,512, fails to provide adequate support or enablement in the manner provided by 35 U.S.C. 112(a) or pre-AIA 35 U.S.C. 112, first paragraph for one or more claims of this application. This application discloses a Cas12i2 domain comprising an amino acid sequence with at least 95% identity to SEQ ID NO: 40 (pg. 77, lines 19-21; Table 3), but the application does not disclose a heterologous sequence comprising an amino acid sequence with at least 88% identity to SEQ ID NO: 61. The application discloses “may comprise a NLS such as an SV40, c-Myc-NLS, or other suitable monopartite NLS” (pg. 27, lines 30-32), and “in some embodiments the NLS is monopartite or bipartite” (pg. 42, lines 9-10). The application further discloses by reference of US2015-0246139, particularly Table 6 (pg. 42, lines 17-19), but an ABSS search alignment of SEQ ID NO: 61 did not show any match results to '139 or its corresponding published US patent 9283287. Further, the teachings of Makkerh et al regarding SEQ ID NO: 61 is discussed above as applied to application ‘651. Therefore, this application fails to provide written description support for a heterologous sequence of SEQ ID NO: 61, and as a result also fails to disclose a Cas12i2 fusion protein of instant claim 1.
Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55. The WO 2022/159585 publication (received on July 19, 2023) of International Patent Application PCT/US2022/013133 discloses SEQ ID NO: 61 and the recitation of a NLS comprising at least 88% identity to SEQ ID NO: 61 (pg. 50, lines 16-22; pg. 129). WO ‘585 further disclose a Cas12i2 fusion protein comprising a Cas12i2 domain comprising an amino acid sequence with at least 95% identity to SEQ ID NO: 40, with recited mutations at specific positions (pg. 86, lines 29-30; pg. 118; pg. 12, lines 30-33).
Accordingly, claims 50-53, 54-67 have an effective filling date of January 20, 2022, which is the filling date of PCT/US2022/013133.
Specification
The disclosure is objected to because it contains an embedded hyperlink (pg. 48, line 31) and/or other form of browser-executable code. 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.
Claim Objections
Claims 50, 62, and 66 are objected to because of the following informalities:
In claim 50, lines 3-4, the recitation of “wherein the Cas12i2 domain comprises an R at position 581, and R at position 926, and a G at position 1030”, but SEQ ID NO: 40 already comprises these amino acid substitutions so it is not necessary to specify them in the claim.
Further, in claim 50, lines 3 and 4, there is recitation of single letters “R” and “G” without definition in the claim. If “R” and “G” are referring to amino acid mutations at specific positions, it is recommended that the claim specifies so. For example, a Cas12i2 domain comprises amino acid substitutions of D581R, I926R, and V1030G.
In claim 62, line 3, there is a recitation of “(ii)” but there is no prior recitation of “(i)” in the claim. It is recommended to amend the claim, lines 2-3, to recite “delivering to the target nucleic acid, (i) a Cas12i2 fusion protein…, and (ii) an RNA guide”.
In claim 66, line 2, the acronym “NLS” is recited, but not defined in the claim. An acronym should be defined the first time it appears in an independent claim or in the group of claims under an independent claim. For the purposes of examination, “NLS” is interpreted to mean “nuclear localization sequence”, as defined in the specification (pg. 50, line 6).
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.
Claim 62 is 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 62 recites the limitation "the CRISPR-associated protein" in line 6. There is insufficient antecedent basis for this limitation in the claim. There is no prior recitation of “CRISPR-associated protein”. Thus, it is unclear which element is being further defined in the instant claim.
Claim Rejections - 35 USC § 112
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 58, 61, and 64-65 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.
Claim 58 is drawn to a nucleic acid encoding the Cas12i2 fusion protein of claim 50; however, claim 50 requires a Cas12i2 fusion protein which is absent in claim 58. Thus, claim 58 fails to include all of the limitations of the claim from which it depends.
Claim 61 is drawn to a composition comprising a first nucleic acid encoding the Cas12i2 fusion protein of claim 50; however, claim 50 requires a Cas12i2 fusion protein which is absent in claim 61. Thus, claim 61 fails to include all of the limitations of the claim from which it depends.
Claim 64 is drawn to a nucleic acid encoding the system of claim 63; however, claim 63 requires a Cas12i2 fusion protein and an RNA, which are absent in claim 64. Thus, claim 61 fails to include all of the limitations of the claim from which it depends.
Claim 65 is drawn to a cell comprising the system of claim 63; however, claim 63 requires a Cas12i2 fusion protein and an RNA or nucleic acid encoding the RNA, which are not required in claim 65. Thus, claim 65 fails to include all of the limitations of the claim from which it depends.
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 50-52, 54-66 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.
MPEP 2163.II.A.3.(a).i) states, “Whether the specification shows that applicant was in possession of the claimed invention is not a single, simple determination, but rather is a factual determination reached by considering a number of factors. Factors to be considered in determining whether there is sufficient evidence of possession include the level of skill and knowledge in the art, partial structure, physical and/or chemical properties, functional characteristics alone or coupled with a known or disclosed correlation between structure and function, and the method of making the claimed invention”.
In making a determination of whether the application complies with the written description requirement of 35 U.S.C. 112, first paragraph, it is necessary to understand what Applicant has possession of and what Applicant is claiming. Claim 50 is drawn to a Cas12i2 domain comprising an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 40. The specification discloses “the term “Cas12i2 fusion protein” refers to…wherein the Cas12i2 fusion protein comes into contact with a target nucleic acid specified by an RNA guide” ([0129]). Therefore, the breadth of the claims encompasses a genus of Cas12i2 domains defined by both structural and functional characteristics, namely domains comprising at least 95% sequence identity to SEQ ID NO: 40 and able to contact a target nucleic acid through interaction with an RNA guide.
SEQ ID NO: 40 consists of 1,054 amino acids, specifically arginine at positions 581 and 926 and valine at position 1030. Accordingly, 1,051 amino acids of SEQ ID NO: 40 can be varied. The claimed minimum sequence identity threshold allows variation up to 52 amino acids (5% of 1,051 is 52.55, rounded to 52) while satisfying the requirement of at least 95% sequence identity to SEQ ID NO: 40. Therefore, the claimed genus encompasses an enormous number of species comprising up to 52 amino acid substitutions, insertions, and deletions, while maintaining the required functional characteristic.
The specification discloses eleven examples identifying locations within the Cas12i2 domain at which a nuclear localization sequence (NLS) may be inserted (pg. 116) and seven circularly permuted Cas12i2 domains reported to retain the ability to interact with a target nucleic acid and perform gene editing (FIG. 14; Example 2). The specification further discloses single amino acid substitutions at every position relative to wild type Cas12i2, SEQ ID NO: 1 (Table 2; pg. 61, lines 13-14), and combinations of up to 8 amino acid substitutions also relative to wild type Cas12i2 (Table 3, pg. 86) where SEQ ID NO: 40 already comprises 3 of those substitutions.
These disclosures do not constitute a representative number of species across the claimed genus, and there is no guidance regarding which substitutions may be combined, except those specified in Table 3, the extent to which the substitutions may be accumulated, or whether the particular combinations would preserve the claimed function. Further, although there is guidance of NLS insertions within the Cas12i2 domain, instantly claimed SEQ ID NO: 61 is only 16 amino acids long while claimed genus encompasses species with modifications of up to 52 amino acids. There is also no guidance whether deletions can be modified into the Cas12i2 domain of SEQ ID NO: 4 while preserving the claimed function. Therefore, the specification does not provide sufficient disclosure of what the complete structure would be of any Cas12i2 domains comprising at least 95% identity to SEQ ID NO: 4.
Prior art studies on mutagenesis resulting in functional Cas proteins appears to be through trial and error. For example, Slaymaker et al (Rationally engineered Cas9 nucleases with improved specificity; Science, 2016, 351(6268): 84-88) teaches methods of mutating Cas9 using a rational mutagenesis to reduce off-target cleavage. Slaymaker, using knowledge of the crystal structure of the Cas9 in contact with target DNA, suspected that non-specific cleavage was a result of positively charged amino acids within the binding pocket, and then mutated 31 positively charged amino acids within the binding pocket to alanine, in order to determine if off-target cleavage was reduced while maintaining target cleavage (See page 84). Each mutant was tested for cleavage of target DNA, as well as known off-target sites. While some of the 31 were capable of maintaining target DNA cleavage, some mutations increased cleavage at off-site locations compared to WT (K862A, K1296A, H1296A and K1300A). Further, a K974A mutation appeared to abolish all cleavage, including cleavage of target DNA (Fig. S3, attached below). Thus, Slaymaker shows rational mutagenesis can be used to target specific residues for mutagenesis, but the functional result requires testing and is unpredictable.
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Further, Strecker et al (Engineering of CRISPR-12b for Human Genome Editing; Nature Communications, 2019, 10 (212): 1-8) relied on the crystalized structure of Cas12b interacting with target DNA, identified 12 amino acids residues within the DNA interacting pocket, and generated Cas12b variants bearing 176 single amino acid substitutions (pg. 2, Fig. 2b-2e). In testing cleavage of VEGFA and DNMT1, of the 176 variants, Strecker shows many of the variants have reduced cleavage VEGF compared to wildtype (Fig. 2C); thus, mutations at different residues identified by rational engineering can also have different functional effected when tested.
Further, Huang et al (Structural Basis for Two Metal-Ion Catalysis of DNA Cleavage by Cas12i2; Nature Communications, 2020, 11(5241): 1-14) also relied on crystalized structure of Cas12i2 interacting with target DNA, identifies amino acid residues within the DNA interacting pocket, and generates single alanine amino acid substitutions at positions considered to have specific interactions with the crRNA repeat region, interactions with dsRNA or the RNA:DNA duplex, or the PAM sequence, in an attempt to identify residues required for Cas12i2 function (pages 2, 4, 6, Fig. 2d, Fig. 3d, Supplemental Fig. 2c-2e). In these alanine variants, Huang et al shows some of the variants have no effect on cleavage. For example, Huang shows the mutations made within the crRNA repeat binding region had little to no effect (Supplemental Fig. 2c), mutations within the RNA:DNA duplex interaction appear to have reduced cleavage, but only one mutation is capable of abolishing all cleavage (Fig. 2d)m and one of the variants with mutations within the PAM interacting domain does not appear to reduce cleavage either (Fig. 3d). Thus, Huang et al also supports that Cas12i2 mutants generated by rational engineering is unpredictable and requires testing to validate mutants’ function.
Further, dependent claims 52 and 54-66 are also rejected for depending from a rejected claim and failing to remedy the lack of written description therein. The breadth of claims 51 and 52 also encompasses subgenus comprising numerous species of Cas12i2 domains with at least 98% and 99% identity to SEQ ID NO: 40, respectively, which are Cas12i2 variants that tolerate at least 10 amino acid mutations. The specification only provides sufficient guidance for up to 8 amino acid substitutions (Table 3, pg. 86) and prior art demonstrates even a single amino acid substitution can result in unpredicted and nonfunctional Cas variants.
On the contrary, dependent claim 67 is drawn to a specific Cas12i2 domain comprising an amino acid sequence of 100% identity to SEQ ID NO: 40, which the specification provides sufficient written description support.
Based on the preponderance of the evidence, including the relevant teachings of the specification, the absence of working examples, and the state of prior art including the knowledge of engineering Cas proteins, one skilled in the art would conclude that Applicant was not in possession of the claimed genus of Cas12i2 domains with at least 95% sequence identity to SEQ ID NO: 40.
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 50-52, 54-67 are rejected under 35 U.S.C. 103 as being unpatentable over Chong et al (WO 2021/202800 A1; Published Date: Oct 07, 2021) in view of Cheng et al (US 2020/0063126 A1; Published Date: Feb 27, 2020).
Regarding claim 50-52, 54, 56-57, 66, and 67, Chong et al teach a Cas12i2 fusion protein comprising:
(a) a Cas12i2 domain comprising an amino acid sequence of SEQ ID NO: 4, which is 100% identical to instant SEQ ID NO: 40, wherein the Cas12i2d domain comprises an arginine at positions 581 and 926, and a glycine at position 1030; and
(b) a heterologous sequence comprising a fusion domain comprising at least one nuclear localization signal (NLS) (pg. 69, para. 2).
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However, Chong et al do not teach wherein the fusion domain comprises an amino acid sequence with at least 88% identity to SEQ ID NO: 61.
Cheng et al teach a Cas12i2 fusion protein comprising:
(a) a Cas12i2 domain comprising an amino acid sequence of SEQ ID NO: 5, which is 99.6% identical to instant SEQ ID NO: 40, comprising amino acid substitutions of an arginine at positions 581 and 926, and a valine at position 1030 (pg. 60-61); and
(b) a heterologous sequence comprising a fusion domain comprising an amino acid sequence of SEQ ID NO: 301, which is 100% identical to instant SEQ ID NO: 61 (i.e., KRPAATKKAGQAKKKK). Cheng et al further teach at least one NLS is attached to the N-terminal and/or C-terminal of the Cas12i2 domain for “optimal expression and nuclear targeting in eukaryotic cells, e.g., human cells” ([0219]).
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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 NLS sequence of the Cas12i2 fusion protein of Chong et al with the NLS of Cheng 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 (NLS) and its function (facilitate delivery of Cas12i2 domain into nucleus of eukaryotic cells) were known in the art. This substitution is merely swapping similar features that serve the same purpose. One would have been motivated to have done so for the advantage of optimizing expression of Cas12i2 fusion protein and nuclear targeting in eukaryotic cells taught by Cheng et al. One would have had a reasonable expectation of success in doing so because Cheng et al already teach a Cas12i2 fusion protein comprising the NLS of SEQ ID NO: 61 and a Cas12i2 domain that is only one amino acid difference to SEQ ID NO: 40.
Regarding claim 55, Chong et al teach the Cas12i2 fusion protein comprises at least one heterologous sequence.
However, Chong et al do not teach wherein the heterologous sequence further comprises at least one linker sequence.
Cheng et al teach a variety of linkers that connect the Cas12i2 domain to an appended domain, e.g., localization factors, and these linkers include, but are not limited to, flexible glycine-serine linkers in various combinations and lengths, rigid linkers, as well as different combinations thereof listed in Table 11 ([0405]-[0406]).
The obviousness to modify the NLS sequence of Chong et al to instant SEQ ID NO: 61 taught by Cheng et al is discussed above as applied to claim 50. Based on this modification, it would have also been obvious to have implemented the linker sequences taught by Cheng et al to connect the NLS to Cas12i2 domain because it would have merely amounted to a simple combination of prior art elements according to known elements to yield predictable results. One would have been motivated to have done so for the advantage of obtaining Cas12i2 fusion proteins with optimal activity through the usage of adequate linkers. One would have had a reasonable expectation of success in doing so because Cheng et al teach numerous linker sequences suitable for use in Cas12i2 fusion proteins comprising a Cas12i2 domain and NLS.
Regarding claim 58-60, Chong et al teach a vector or nucleic acid encoding the Cas12i2 fusion protein in a cell (pg. 164, para. 3).
Regarding claim 61, Chong et al teach a composition comprising a Cas12i2 fusion protein and an RNA guide that comprises a direct repeat sequence and a spacer sequence (pg. 1, lines 32-34), wherein the spacer sequence is capable of hybridizing to a target nucleic acid (pg. 2, line 9). Chong et al further teach a cell transfected with one or more nucleic acids encoding the Cas12i2 fusion protein and RNA guide (i.e., a first and a second nucleic acid) (pg. 181, para. 2).
Regarding claims 62-65, Chong et al teach a method of introducing a deletion in a cell (i.e., modifying a target nucleic acid) by delivering the composition (i.e., system) (pg. 163, para. 4) comprising: i) a Cas12i2 fusion protein or a nucleic acid encoding the Cas12i2 fusion protein; and ii) an RNA guide comprising a direct repeat sequence and a spacer sequence (pg. 1, lines 32-34), wherein the spacer sequence is capable of hybridizing to the target nucleic acid (pg. 2, line 9). Chong et al further teach the Cas12i2 fusion protein and the RNA guide form a binary complex, in which the RNA guide exhibits decreased dissociation (i.e., the Cas12i2 fusion protein is capable of binding to the RNA guide) (pg. 4, para. 2). Chong et al further teach the Cas12i2 fusion protein contacts with the DNA of the cell, resulting in modification of the target DNA due to non-homologous end joining or homologous-directed repair (i.e., wherein recognition of the target nucleic acid by the Cas12i2 fusion protein and the RNA guide results in a modification of the target nucleic acid) (pg. paragraph bridging pg. 181 and pg. 182). Chong et al further teach a vector or nucleic acid encoding the Cas12i2 fusion protein and the RNA guide is transfected into a cell (pg. 164, para. 3; pg. 13, lines 22-23).
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 50-52, 54-67 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 3-4, 7, 10-11, 17, and 20 of U.S. Patent No. 11939607 in view of Cheng et al (US 2020/0063126 A1; Published Date: Feb 27, 2020).
Regarding instant claims 50-52, 54-67, ‘607 teaches a Cas12i2 domain of SEQ ID NO: 1168, which is 100 identical to instant SEQ ID NO: 40, comprising an arginine at positions 581 and 926 and a glycine at position 1030 (claim 3). ‘607 further teach a system or composition comprising nucleic acids encoding for the Cas12i2 domain and an RNA guide (i.e., first and second nucleic acid), wherein the RNA comprises a direct repeat sequence and a spacer sequence that binds to a target nucleic acid, and wherein the Cas12i2 domain binds to the RNA guide (claims 1, 7, and 17). ‘245 further teach using this system to edit a lactate dehydrogenase A gene in a cell (i.e., modifying a target nucleic acid) (claim 62).
Alignment of instant SEQ ID NO: 40 and SEQ ID NO: 1168 of ‘607
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However, ‘607 does not teach wherein the Cas12i2 domain is fused to a nuclear localization sequence (NLS) comprising an amino acid sequence with at least 81% identity to SEQ ID NO: 61, and wherein the NLS further comprises at least one linker sequence.
Cheng et al teach a Cas12i2 fusion protein comprising: (a) a Cas12i2 domain comprising an amino acid sequence of SEQ ID NO: 5, which is 99.6% identical to instant SEQ ID NO: 40, comprising amino acid substitutions of an arginine at positions 581 and 926, and a valine at position 1030 (pg. 60-61); and (b) a heterologous sequence comprising a fusion domain comprising an amino acid sequence of SEQ ID NO: 301, which is 100% identical to instant SEQ ID NO: 61 (i.e., KRPAATKKAGQAKKKK). Cheng et al further teach at least one NLS is attached to the N-terminal and/or C-terminal of the Cas12i2 domain for “optimal expression and nuclear targeting in eukaryotic cells, e.g., human cells” ([0219]). Cheng et al also teach a variety of linkers that connect the Cas12i2 domain to an appended domain, e.g., localization factors, and these linkers include, but are not limited to, flexible glycine-serine linkers in various combinations and lengths, rigid linkers, as well as different combinations thereof listed in Table 11 ([0405]-[0406]).
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 Cas12i2 domain of ‘607 by fusing to at least one NLS taught by Cheng et al because it would have merely amounted to a simple combination of prior art elements according to known methods to yield predictable results. Each element in the combination (Cas12i2 domain and NLS) merely performs the same function as it does separately (Cas12i2 domain interacts with an RNA guide to perform gene editing and NLS directs entry of the Cas12i2 domain into the nucleus). One would have been motivated to have done so for the advantage of optimizing expression of Cas12i2 fusion protein and nuclear targeting in eukaryotic cells taught by Cheng et al. One would have had a reasonable expectation of success in doing so because Cheng et al already teach a Cas12i2 fusion protein comprising the NLS of SEQ ID NO: 61 and a Cas12i2 domain that is only one amino acid difference to SEQ ID NO: 40.
Claims 50-52, 54-67 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 2, 10, 13-14, and 20 of U.S. Patent No. 11821012 in view of Cheng et al (US 2020/0063126 A1; Published Date: Feb 27, 2020).
Regarding instant claims 50-52, 54-67, ‘012 teaches a Cas12i2 domain that is a variant of SEQ ID NO: 922 (98.8% identity to instant SEQ ID NO: 40) and ‘012 teaches the Cas12i2 domain variant comprises instantly required mutations of D581R, I926R, and V1030R (claim 2). ‘012 further teach a composition or system comprising: i) a first nucleic acid encoding the Cas12i2 domain; and ii) a second nucleic acid encoding an RNA guide comprising direct repeat sequence and a spacer sequence specific to a target nucleic acid (claims 20, 10, 13 and 14). ‘012 further teach the Cas12i2 domain and the RNA guide in combination performs genetic editing of a hydroxy-acid oxidase 1 (i.e., modifying a target nucleic acid) (claim 1).
Alignment of instant SEQ ID NO: 40 and SEQ ID NO: 922 of ‘012
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However, ‘012 does not teach the Cas12i2 domain is fused to a heterologous sequence comprising i) a fusion domain with at least 88% identity to SEQ ID NO: 61 and ii) at least one linker sequence. ‘012 does not teach a cell comprising the Cas12i2 fusion protein.
Cheng et al teach a Cas12i2 fusion protein comprising: (a) a Cas12i2 domain comprising an amino acid sequence of SEQ ID NO: 5, which is 99.6% identical to instant SEQ ID NO: 40, comprising amino acid substitutions of an arginine at positions 581 and 926, and a valine at position 1030 (pg. 60-61); and (b) a heterologous sequence comprising a fusion domain comprising an amino acid sequence of SEQ ID NO: 301, which is 100% identical to instant SEQ ID NO: 61 (i.e., KRPAATKKAGQAKKKK). Cheng et al further teach at least one NLS is attached to the N-terminal and/or C-terminal of the Cas12i2 domain for “optimal expression and nuclear targeting in eukaryotic cells, e.g., human cells” ([0219]). Cheng et al also teach a variety of linkers that connect the Cas12i2 domain to an appended domain, e.g., localization factors, and these linkers include, but are not limited to, flexible glycine-serine linkers in various combinations and lengths, rigid linkers, as well as different combinations thereof listed in Table 11 ([0405]-[0406]).
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 Cas12i2 domain of ‘012 by fusing to at least one NLS taught by Cheng et al because it would have merely amounted to a simple combination of prior art elements according to known methods to yield predictable results. Each element in the combination (Cas12i2 domain and NLS) merely performs the same function as it does separately (Cas12i2 domain interacts with an RNA guide to perform gene editing and NLS directs entry of the Cas12i2 domain into the nucleus). One would have been motivated to have done so for the advantage of optimizing expression of Cas12i2 fusion protein and nuclear targeting in eukaryotic cells taught by Cheng et al. One would have had a reasonable expectation of success in doing so because Cheng et al already teach a Cas12i2 fusion protein comprising the NLS of SEQ ID NO: 61 and a Cas12i2 domain that is only one amino acid difference to SEQ ID NO: 40.
Claim 50-52, 54-67 are rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1, 12, 14, 16, and 19-20 of U.S. Patent No. 10,808,245 in view of Chong et al (WO 2021/202800 A1; Published Date: Oct 07, 2021) and Cheng et al (US 2020/0063126 A1; Published Date: Feb 27, 2020).
Regarding instant claims 50-52, 54-67, ‘245 teaches a Cas12i2 fusion protein comprising: (a) a CRISPR-Cas effector protein (i.e., Cas12i2 domain) of SEQ ID NO: 5, which is 99.6% identical to instant SEQ ID NO: 40, comprising an arginine at positions 581 and 926 but a valine at position 1030; and (b) a heterologous sequence comprising of at least one nuclear localization signal (NLS) (claim 12). ‘245 further teach a system or composition comprising nucleic acids encoding for the Cas12i2 domain and an RNA guide (i.e., first and second nucleic acid), wherein the RNA comprises a direct repeat sequence and a spacer sequence that binds to a target nucleic acid, and wherein the Cas12i2 domain binds to the RNA guide (claim 1). ‘245 further teach a vector comprising the nucleic acid of the Cas12i2 fusion protein (claim 16), and a cell comprising the Cas12i2 fusion protein (claims 14 and 19).
Alignment of instant SEQ ID NO: 40 and SEQ ID NO: 5 of ‘245
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However, ‘245 does not teach wherein the Cas12i2 domain comprises a glycine at position 1030.
Chong et al teach a Cas12i2 fusion protein comprising: (a) a Cas12i2 domain comprising an amino acid sequence of SEQ ID NO: 4, which is 100% identical to instant SEQ ID NO: 40, wherein the Cas12i2 domain comprises an arginine at positions 581 and 926, and a glycine at position 1030; and
(b) a heterologous sequence comprising a fusion domain comprising at least one nuclear localization signal (NLS) (pg. 69, para. 2). Chong et al further teach mutations in SEQ ID NO: 4 exhibits increased stability and/or protein-DNA interactions, specifically V1030G is at the C-terminal portion of the Cas12i2 domain (FIG. 20B, pg. 34, lines 1-3). Chong et al also teach using this Cas12i2 fusion protein in a method of introducing a deletion in a cell (i.e., modifying a target nucleic acid) by delivering the composition (i.e., system) (pg. 163, para. 4) comprising: i) a Cas12i2 fusion protein or a nucleic acid encoding the Cas12i2 fusion protein; and ii) an RNA guide comprising a direct repeat sequence and a spacer sequence (pg. 1, lines 32-34).
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 Cas12i2 domain of ‘245 to comprise mutation V1030G taught by Chong et al because it would have merely amounted to a simple substitution of prior art elements according to known methods to yield predictable results. One would have been motivated to have done so for the advantage of obtaining a Cas12i2 variant with increased stability and capable of forming binary complexes with decreased dissociations with RNA guides, as well as modifying a target nucleic acid using this Cas12i2 variant of Chong et al. One would have had a reasonable expectation of success in doing so because Chong et al already teach a Cas12i2 domain that is only one amino acid difference to SEQ ID NO: 5 of ‘245.
However, ‘245 does not teach wherein the NLS is at least 88% identical to SEQ ID NO: 61.
Cheng et al teach a Cas12i2 fusion protein comprising: (a) a Cas12i2 domain comprising an amino acid sequence of SEQ ID NO: 5, which is 99.6% identical to instant SEQ ID NO: 40, comprising amino acid substitutions of an arginine at positions 581 and 926, and a valine at position 1030 (pg. 60-61); and (b) a heterologous sequence comprising a fusion domain comprising an amino acid sequence of SEQ ID NO: 301, which is 100% identical to instant SEQ ID NO: 61 (i.e., KRPAATKKAGQAKKKK). Cheng et al further teach at least one NLS is attached to the N-terminal and/or C-terminal of the Cas12i2 domain for “optimal expression and nuclear targeting in eukaryotic cells, e.g., human cells” ([0219]). Cheng et al also teach a variety of linkers that connect the Cas12i2 domain to an appended domain, e.g., localization factors, and these linkers include, but are not limited to, flexible glycine-serine linkers in various combinations and lengths, rigid linkers, as well as different combinations thereof listed in Table 11 ([0405]-[0406]).
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 Cas12i2 domain of ‘245 by fusing to at least one NLS taught by Cheng et al because it would have merely amounted to a simple combination of prior art elements according to known methods to yield predictable results. Each element in the combination (Cas12i2 domain and NLS) merely performs the same function as it does separately (Cas12i2 domain interacts with an RNA guide to perform gene editing and NLS directs entry of the Cas12i2 domain into the nucleus). One would have been motivated to have done so for the advantage of optimizing expression of Cas12i2 fusion protein and nuclear targeting in eukaryotic cells taught by Cheng et al. One would have had a reasonable expectation of success in doing so because Cheng et al already teach a Cas12i2 fusion protein comprising the NLS of SEQ ID NO: 61 and a Cas12i2 domain that is only one amino acid difference to SEQ ID NO: 40.
Claims 50-52, 54-67 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 7, 13, 21, 25, 28, 62, 64, 70, and 89 of copending Application No. 17/830,212 in view of Cheng et al (US 2020/0063126 A1; Published Date: Feb 27, 2020).
Regarding instant claims 50-52, 54-67, ‘212 teach a Cas12i2 fusion protein comprising: (a) a Type V CRISPR nuclease polypeptide (i.e., Cas12i2 domain) of SEQ ID NO: 4, which is 100% identical to instant SEQ ID NO: 40, comprising an arginine at positions 581 and 926 and a glycine at position 1030 (claims 7 and 89); and (b) a heterologous sequence comprising of a reverse transcriptase (claim 21). ‘212 further teach a system or composition comprising nucleic acids encoding for the Cas12i2 domain and an RNA guide (i.e., first and second nucleic acid), wherein the RNA comprises a direct repeat sequence and a spacer sequence that binds to a target nucleic acid, and wherein the Cas12i2 domain binds to the RNA guide (claim 1, 13, 25, 28, 62). ‘212 further teach a method of genetically editing a cell (i.e., modifying a target nucleic acid) using the recited composition (claims 64 and 70).
‘212 further teach a vector comprising the nucleic acid of the Cas12i2 fusion protein (claim 16), and a cell comprising the Cas12i2 fusion protein (claims 14 and 19).
Alignment of instant SEQ ID NO: 40 and SEQ ID NO: 4 of ‘212
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However, ‘212 does not teach the Cas12i2 domain is fused to a heterologous sequence comprising i) a fusion domain with at least 88% identity to SEQ ID NO: 61 and ii) at least one linker sequence. ‘212 does not teach a cell comprising the Cas12i2 fusion protein.
Cheng et al teach a Cas12i2 fusion protein comprising: (a) a Cas12i2 domain comprising an amino acid sequence of SEQ ID NO: 5, which is 99.6% identical to instant SEQ ID NO: 40, comprising amino acid substitutions of an arginine at positions 581 and 926, and a valine at position 1030 (pg. 60-61); and (b) a heterologous sequence comprising a fusion domain comprising an amino acid sequence of SEQ ID NO: 301, which is 100% identical to instant SEQ ID NO: 61 (i.e., KRPAATKKAGQAKKKK). Cheng et al further teach at least one NLS is attached to the N-terminal and/or C-terminal of the Cas12i2 domain for “optimal expression and nuclear targeting in eukaryotic cells, e.g., human cells” ([0219]). Cheng et al also teach a variety of linkers that connect the Cas12i2 domain to an appended domain, e.g., localization factors, and these linkers include, but are not limited to, flexible glycine-serine linkers in various combinations and lengths, rigid linkers, as well as different combinations thereof listed in Table 11 ([0405]-[0406]).
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 Cas12i2 domain of ‘212 by fusing to at least one NLS taught by Cheng et al because it would have merely amounted to a simple combination of prior art elements according to known methods to yield predictable results. Each element in the combination (Cas12i2 domain and NLS) merely performs the same function as it does separately (Cas12i2 domain interacts with an RNA guide to perform gene editing and NLS directs entry of the Cas12i2 domain into the nucleus). One would have been motivated to have done so for the advantage of optimizing expression of Cas12i2 fusion protein and nuclear targeting in eukaryotic cells taught by Cheng et al. One would have had a reasonable expectation of success in doing so because Cheng et al already teach a Cas12i2 fusion protein comprising the NLS of SEQ ID NO: 61 and a Cas12i2 domain that is only one amino acid difference to SEQ ID NO: 40.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Claims 50-52, 54-67 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 3-4, 13-14, 17-18, 233, 411, and 414-415 of copending Application No. 17/916,270 in view of Cheng et al (US 2020/0063126 A1; Published Date: Feb 27, 2020).
Regarding instant claims 50-52, 54-67, ‘270 teaches a Cas12i2 fusion protein comprising: (a) a Cas12i2 domain of SEQ ID NO: 4, which is 100% identical to instant SEQ ID NO: 40, comprising an arginine at positions 581 and 926 and a glycine at position 1030 (claims 411 and 4); and (b) a heterologous sequence comprising of at least one nuclear localization signal (NLS) (claims 14 and 415). ‘270 further teach a system or composition comprising nucleic acids encoding for the Cas12i2 domain and an RNA guide (i.e., first and second nucleic acid), wherein the RNA comprises a direct repeat sequence and a spacer sequence that binds to a target nucleic acid, and wherein the Cas12i2 domain binds to the RNA guide (claims 13, 414). ‘270 further teach a vector or nucleic acid encoding the Cas12i2 fusion protein (claim 17), and a cell comprising the Cas12i2 fusion protein (claim 18). ‘270 further teach a method of obtaining deletion a cell (i.e., modifying a target nucleic acid) using the recited composition (claim 233).
Alignment of instant SEQ ID NO: 40 and SEQ ID NO: 4 of ‘270
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However, ‘270 does not teach the Cas12i2 domain is fused to a heterologous sequence comprising i) a fusion domain with at least 88% identity to SEQ ID NO: 61 and ii) at least one linker.
Cheng et al teach a Cas12i2 fusion protein comprising: (a) a Cas12i2 domain comprising an amino acid sequence of SEQ ID NO: 5, which is 99.6% identical to instant SEQ ID NO: 40, comprising amino acid substitutions of an arginine at positions 581 and 926, and a valine at position 1030 (pg. 60-61); and (b) a heterologous sequence comprising a fusion domain comprising an amino acid sequence of SEQ ID NO: 301, which is 100% identical to instant SEQ ID NO: 61 (i.e., KRPAATKKAGQAKKKK). Cheng et al further teach at least one NLS is attached to the N-terminal and/or C-terminal of the Cas12i2 domain for “optimal expression and nuclear targeting in eukaryotic cells, e.g., human cells” ([0219]). Cheng et al also teach a variety of linkers that connect the Cas12i2 domain to an appended domain, e.g., localization factors, and these linkers include, but are not limited to, flexible glycine-serine linkers in various combinations and lengths, rigid linkers, as well as different combinations thereof listed in Table 11 ([0405]-[0406]).
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 Cas12i2 domain of ‘270 by fusing to at least one NLS of SEQ ID NO: 61 via optimized linkers taught by Cheng et al because it would have merely amounted to a simple combination of prior art elements according to known methods to yield predictable results. Each element in the combination (Cas12i2 domain and NLS) merely performs the same function as it does separately (Cas12i2 domain interacts with an RNA guide to perform gene editing and NLS directs entry of the Cas12i2 domain into the nucleus). One would have been motivated to have done so for the advantage of optimizing expression of Cas12i2 fusion protein and nuclear targeting in eukaryotic cells taught by Cheng et al. One would have had a reasonable expectation of success in doing so because Cheng et al already teach a Cas12i2 fusion protein comprising the NLS of SEQ ID NO: 61 and a Cas12i2 domain that is only one amino acid difference to SEQ ID NO: 40.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Claims 50-52, 54-67 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 4, 6-7, 13, 19, 21, 27, and 49 of copending Application No. 17/832,114 in view of Chong et al (WO 2021/202800 A1; Published Date: Oct 07, 2021) and Cheng et al (US 2020/0063126 A1; Published Date: Feb 27, 2020).
Regarding instant claims 50-52, 54-67, ‘114 teaches a Cas12i2 domain of SEQ ID NO: 1166, which is 99.6% identical to instant SEQ ID NO: 40, comprising an arginine at positions 581 and 926 but a valine at position 1030 (claim 4). ‘114 further teach a system or composition comprising nucleic acids encoding for the Cas12i2 domain and an RNA guide (i.e., first and second nucleic acid), wherein the RNA comprises a direct repeat sequence and a spacer sequence that binds to a target nucleic acid, and wherein the Cas12i2 domain binds to the RNA guide (claims 6, 13, 19, and 21). ‘114 further teach a vector comprising the nucleic acid of the Cas12i2 fusion protein (claim 7), and a cell comprising the Cas12i2 fusion protein (claim 49). ‘114 further teach a method of genetically editing a cell (i.e., modifying a target nucleic acid) using the recited composition (claims 27 and 49).
Alignment of instant SEQ ID NO: 40 and SEQ ID NO: 1166 of ‘114
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However, ‘114 does not teach wherein the Cas12i2 domain comprises a glycine at position 1030.
Chong et al teach a Cas12i2 fusion protein comprising: (a) a Cas12i2 domain comprising an amino acid sequence of SEQ ID NO: 4, which is 100% identical to instant SEQ ID NO: 40, wherein the Cas12i2 domain comprises an arginine at positions 581 and 926, and a glycine at position 1030; and (b) a heterologous sequence comprising a fusion domain comprising at least one nuclear localization signal (NLS) (pg. 69, para. 2). Chong et al further teach mutations in SEQ ID NO: 4 exhibits increased stability and/or protein-DNA interactions, specifically V1030G is at the C-terminal portion of the Cas12i2 domain (FIG. 20B, pg. 34, lines 1-3).
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 Cas12i2 domain of ‘114 to comprise mutation V1030G taught by Chong et al because it would have merely amounted to a simple substitution of prior art elements according to known methods to yield predictable results. One would have been motivated to have done so for the advantage of obtaining a Cas12i2 variant with increased stability and capable of forming binary complexes with decreased dissociations with RNA guides, as well as modifying a target nucleic acid using this Cas12i2 variant of Chong et al. One would have had a reasonable expectation of success in doing so because Chong et al already teach a Cas12i2 domain that is only one amino acid difference to SEQ ID NO: 1166 of ‘114.
However, ‘114 does not teach the Cas12i2 domain is fused to a heterologous sequence comprising i) a fusion domain with at least 88% identity to SEQ ID NO: 61 and ii) at least one linker sequence. ‘012 does not teach a cell comprising the Cas12i2 fusion protein.
Cheng et al teach a Cas12i2 fusion protein comprising: (a) a Cas12i2 domain comprising an amino acid sequence of SEQ ID NO: 5, which is 99.6% identical to instant SEQ ID NO: 40, comprising amino acid substitutions of an arginine at positions 581 and 926, and a valine at position 1030 (pg. 60-61); and (b) a heterologous sequence comprising a fusion domain comprising an amino acid sequence of SEQ ID NO: 301, which is 100% identical to instant SEQ ID NO: 61 (i.e., KRPAATKKAGQAKKKK). Cheng et al further teach at least one NLS is attached to the N-terminal and/or C-terminal of the Cas12i2 domain for “optimal expression and nuclear targeting in eukaryotic cells, e.g., human cells” ([0219]). Cheng et al also teach a variety of linkers that connect the Cas12i2 domain to an appended domain, e.g., localization factors, and these linkers include, but are not limited to, flexible glycine-serine linkers in various combinations and lengths, rigid linkers, as well as different combinations thereof listed in Table 11 ([0405]-[0406]).
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 Cas12i2 domain of ‘114 by fusing to at least one NLS of SEQ ID NO: 61 via optimized linkers taught by Cheng et al because it would have merely amounted to a simple combination of prior art elements according to known methods to yield predictable results. Each element in the combination (Cas12i2 domain and NLS) merely performs the same function as it does separately (Cas12i2 domain interacts with an RNA guide to perform gene editing and NLS directs entry of the Cas12i2 domain into the nucleus). One would have been motivated to have done so for the advantage of optimizing expression of Cas12i2 fusion protein and nuclear targeting in eukaryotic cells taught by Cheng et al. One would have had a reasonable expectation of success in doing so because Cheng et al already teach a Cas12i2 fusion protein comprising the NLS of SEQ ID NO: 61 and a Cas12i2 domain that is only one amino acid difference to SEQ ID NO: 40.
This is a provisional nonstatutory double patenting rejection.
Claims 50-52, 54-67 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1, 15, 37, 46, 49, 51, 54, and 54 of copending Application No. 17/931,027 in view of Cheng et al (US 2020/0063126 A1; Published Date: Feb 27, 2020).
Regarding instant claims 50-52, 54-67, ‘027 teaches a Cas12i2 fusion protein comprising a Cas12i2 domain of SEQ ID NO: 2, which is 99.6% identical to instant SEQ ID NO: 40, comprising an arginine at positions 581 and 926 but a valine at position 1030. However, ‘027 also teaches a Cas12i2 domain comprising an alteration relative to the amino acid sequence of SEQ ID NO: 2, wherein the alteration comprises V1030G (claim 15). Thus, ‘027 teaches a Cas12i2 domain that is 100% identical to instant SEQ ID NO: 40. ‘027 further reach the Cas12i2 fusion protein comprises at least one heterologous sequence that further comprises at least one linker (claims 37 and 46). ‘027 further teach a composition comprising the Cas12i fusion protein and an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide comprises a nuclease binding sequence (i.e., direct repeat sequence) and a DNA-binding sequence (i.e., a spacer sequence) that is substantially identical to a target nucleic acid (i.e., capable of hybridizing) (claims 54 and 56). ‘027 further teach wherein the Cas12i fusion protein forms a complex with an RNA guide (i.e., wherein the Cas12i2 fusion protein is capable of binding to the RNA guide) (claim 49) and the Cas12i fusion protein is used in a method of introducing a substitution in a target sequence in a cell (i.e., modifying a target nucleic acid) (claim 51)
Alignment of instant SEQ ID NO: 40 and SEQ ID NO: 2 of ‘027
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However, ‘027 does not teach the Cas12i2 domain is fused to a heterologous sequence comprising i) a fusion domain with at least 88% identity to SEQ ID NO: 61 and ii) at least one linker sequence. ‘012 does not teach a cell comprising the Cas12i2 fusion protein.
Cheng et al teach a Cas12i2 fusion protein comprising: (a) a Cas12i2 domain comprising an amino acid sequence of SEQ ID NO: 5, which is 99.6% identical to instant SEQ ID NO: 40, comprising amino acid substitutions of an arginine at positions 581 and 926, and a valine at position 1030 (pg. 60-61); and (b) a heterologous sequence comprising a fusion domain comprising an amino acid sequence of SEQ ID NO: 301, which is 100% identical to instant SEQ ID NO: 61 (i.e., KRPAATKKAGQAKKKK). Cheng et al further teach at least one NLS is attached to the N-terminal and/or C-terminal of the Cas12i2 domain for “optimal expression and nuclear targeting in eukaryotic cells, e.g., human cells” ([0219]). Cheng et al also teach a variety of linkers that connect the Cas12i2 domain to an appended domain, e.g., localization factors, and these linkers include, but are not limited to, flexible glycine-serine linkers in various combinations and lengths, rigid linkers, as well as different combinations thereof listed in Table 11 ([0405]-[0406]).
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 Cas12i2 domain of ‘027 by fusing to at least one NLS of SEQ ID NO: 61 via optimized linkers taught by Cheng et al because it would have merely amounted to a simple combination of prior art elements according to known methods to yield predictable results. Each element in the combination (Cas12i2 domain and NLS) merely performs the same function as it does separately (Cas12i2 domain interacts with an RNA guide to perform gene editing and NLS directs entry of the Cas12i2 domain into the nucleus). One would have been motivated to have done so for the advantage of optimizing expression of Cas12i2 fusion protein and nuclear targeting in eukaryotic cells taught by Cheng et al. One would have had a reasonable expectation of success in doing so because Cheng et al already teach a Cas12i2 fusion protein comprising the NLS of SEQ ID NO: 61 and a Cas12i2 domain that is only one amino acid difference to SEQ ID NO: 40.
This is a provisional nonstatutory double patenting rejection.
Claims 50-52, 54-67 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 20-21, 44, and 48 of copending Application No. 18/251,183 in view of Chong et al (WO 2021/202800 A1; Published Date: Oct 07, 2021) and Cheng et al (US 2020/0063126 A1; Published Date: Feb 27, 2020).
Regarding instant claims 50-52, 54-67, ‘183 teaches a Cas12i2 domain of SEQ ID NO: 2634, which is 99.6% identical to instant SEQ ID NO: 40, comprising an arginine at positions 581 and 926 but a valine at position 1030 (claim 18). ‘183 further teach a composition or system comprising the Cas12i2 domain and an RNA guide, wherein they form a ribonucleoprotein complex (i.e., binds to the RNA guide) (claim 20), wherein RNA guide comprises a direct repeat sequence and a spacer sequence that binds to a target nucleic acid (claim 1). ‘183 further teach a vector comprising the nucleic acids encoding for the Cas12i2 domain and the RNA guide (claim 44), as well as a cell comprising the Cas12i2 domain (claim 21). ‘183 further teach using the composition comprising the Cas12i2 domain and the RNA guide in a method to edit a BCL11A sequence (i.e., modifying a target nucleic acid) (claim 48).
Alignment of instant SEQ ID NO: 40 and SEQ ID NO: 2634 of ‘183
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69
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However, ‘183 does not teach wherein the Cas12i2 domain comprises a glycine at position 1030.
Chong et al teach a Cas12i2 fusion protein comprising: (a) a Cas12i2 domain comprising an amino acid sequence of SEQ ID NO: 4, which is 100% identical to instant SEQ ID NO: 40, wherein the Cas12i2 domain comprises an arginine at positions 581 and 926, and a glycine at position 1030; and (b) a heterologous sequence comprising a fusion domain comprising at least one nuclear localization signal (NLS) (pg. 69, para. 2). Chong et al further teach mutations in SEQ ID NO: 4 exhibits increased stability and/or protein-DNA interactions, specifically V1030G is at the C-terminal portion of the Cas12i2 domain (FIG. 20B, pg. 34, lines 1-3).
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 Cas12i2 domain of ‘183 to comprise mutation V1030G taught by Chong et al because it would have merely amounted to a simple substitution of prior art elements according to known methods to yield predictable results. One would have been motivated to have done so for the advantage of obtaining a Cas12i2 variant with increased stability and capable of forming binary complexes with decreased dissociations with RNA guides, as well as modifying a target nucleic acid using this Cas12i2 variant of Chong et al. One would have had a reasonable expectation of success in doing so because Chong et al already teach a Cas12i2 domain that is only one amino acid difference to SEQ ID NO: 2634 of ‘183.
However, ‘183 does not teach the Cas12i2 domain is fused to a heterologous sequence comprising i) a fusion domain with at least 88% identity to SEQ ID NO: 61 and ii) at least one linker sequence. ‘012 does not teach a cell comprising the Cas12i2 fusion protein.
Cheng et al teach a Cas12i2 fusion protein comprising: (a) a Cas12i2 domain comprising an amino acid sequence of SEQ ID NO: 5, which is 99.6% identical to instant SEQ ID NO: 40, comprising amino acid substitutions of an arginine at positions 581 and 926, and a valine at position 1030 (pg. 60-61); and (b) a heterologous sequence comprising a fusion domain comprising an amino acid sequence of SEQ ID NO: 301, which is 100% identical to instant SEQ ID NO: 61 (i.e., KRPAATKKAGQAKKKK). Cheng et al further teach at least one NLS is attached to the N-terminal and/or C-terminal of the Cas12i2 domain for “optimal expression and nuclear targeting in eukaryotic cells, e.g., human cells” ([0219]). Cheng et al also teach a variety of linkers that connect the Cas12i2 domain to an appended domain, e.g., localization factors, and these linkers include, but are not limited to, flexible glycine-serine linkers in various combinations and lengths, rigid linkers, as well as different combinations thereof listed in Table 11 ([0405]-[0406]).
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 Cas12i2 domain of ‘183 by fusing to at least one NLS of SEQ ID NO: 61 via optimized linkers taught by Cheng et al because it would have merely amounted to a simple combination of prior art elements according to known methods to yield predictable results. Each element in the combination (Cas12i2 domain and NLS) merely performs the same function as it does separately (Cas12i2 domain interacts with an RNA guide to perform gene editing and NLS directs entry of the Cas12i2 domain into the nucleus). One would have been motivated to have done so for the advantage of optimizing expression of Cas12i2 fusion protein and nuclear targeting in eukaryotic cells taught by Cheng et al. One would have had a reasonable expectation of success in doing so because Cheng et al already teach a Cas12i2 fusion protein comprising the NLS of SEQ ID NO: 61 and a Cas12i2 domain that is only one amino acid difference to SEQ ID NO: 40.
This is a provisional nonstatutory double patenting rejection.
Claims 50-52, 54-67 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 6-7, 19, 21, 47, and 49 of copending Application No. 17/832,038 in view of Cheng et al (US 2020/0063126 A1; Published Date: Feb 27, 2020).
Regarding instant claims 50-52, 54-67, ‘038 teaches a Cas12i2 domain of SEQ ID NO: 924, which is 100% identical to instant SEQ ID NO: 40, comprising an arginine at positions 581 and 926 and a glycine at position 1030 (claim 1). ‘038 further teach a system or composition comprising nucleic acids encoding for the Cas12i2 domain and an RNA guide (i.e., first and second nucleic acid), wherein the RNA comprises a direct repeat sequence and a spacer sequence that binds to a target nucleic acid, and wherein the Cas12i2 domain binds to the RNA guide (claims 1 and 47). ‘245 further teach a vector comprising the nucleic acid encoding the Cas12i2 fusion protein (claims 7, 19, and 21). ‘038 further teach a method of genetically editing hydroxyacid oxidase 1 gene a cell (i.e., modifying a target nucleic acid) using the recited composition (claim 49).
However, ‘038 does not teach the Cas12i2 domain is fused to a heterologous sequence comprising i) a fusion domain with at least 88% identity to SEQ ID NO: 61 and ii) at least one linker sequence.
Cheng et al teach a Cas12i2 fusion protein comprising: (a) a Cas12i2 domain comprising an amino acid sequence of SEQ ID NO: 5, which is 99.6% identical to instant SEQ ID NO: 40, comprising amino acid substitutions of an arginine at positions 581 and 926, and a valine at position 1030 (pg. 60-61); and (b) a heterologous sequence comprising a fusion domain comprising an amino acid sequence of SEQ ID NO: 301, which is 100% identical to instant SEQ ID NO: 61 (i.e., KRPAATKKAGQAKKKK). Cheng et al further teach at least one NLS is attached to the N-terminal and/or C-terminal of the Cas12i2 domain for “optimal expression and nuclear targeting in eukaryotic cells, e.g., human cells” ([0219]). Cheng et al also teach a variety of linkers that connect the Cas12i2 domain to an appended domain, e.g., localization factors, and these linkers include, but are not limited to, flexible glycine-serine linkers in various combinations and lengths, rigid linkers, as well as different combinations thereof listed in Table 11 ([0405]-[0406]).
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 Cas12i2 domain of ‘038 by fusing to at least one NLS of SEQ ID NO: 61 via optimized linkers taught by Cheng et al because it would have merely amounted to a simple combination of prior art elements according to known methods to yield predictable results. Each element in the combination (Cas12i2 domain and NLS) merely performs the same function as it does separately (Cas12i2 domain interacts with an RNA guide to perform gene editing and NLS directs entry of the Cas12i2 domain into the nucleus). One would have been motivated to have done so for the advantage of optimizing expression of Cas12i2 fusion protein and nuclear targeting in eukaryotic cells taught by Cheng et al. One would have had a reasonable expectation of success in doing so because Cheng et al already teach a Cas12i2 fusion protein comprising the NLS of SEQ ID NO: 61 and a Cas12i2 domain that is only one amino acid difference to SEQ ID NO: 40.
This is a provisional nonstatutory double patenting rejection.
Claims 50-52, 54-67 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 4, 6, 8, 13, 20-22, 47, 49, and 52 of copending Application No. 18/565,575 in view of Cheng et al (US 2020/0063126 A1; Published Date: Feb 27, 2020).
Regarding instant claims 50-52, 54-67, ‘575 teaches a Cas12i2 domain of SEQ ID NO: 922, which is 99.6% identical to instant SEQ ID NO: 40, comprising an arginine at positions 581 and 926 but a valine at position 1030. However, ‘575 also teaches a Cas12i2 domain comprising mutations relative to the amino acid sequence of SEQ ID NO: 922, wherein one of the mutations comprises V1030G (claim 4). Thus, ‘575 teaches a Cas12i2 domain that is 100% identical to instant SEQ ID NO: 40. ‘575 further teach a system or composition comprising vectors or nucleic acids encoding for the Cas12i2 domain and an RNA guide (i.e., first and second nucleic acid), wherein the RNA comprises a direct repeat sequence and a spacer sequence that binds to a target nucleic acid, and wherein the Cas12i2 domain binds to the RNA guide (claims 1, 6, 8, 13, 20-22, and 47). ‘575 further teach a method of editing a hydroxyacid oxidase 1 gene a cell (i.e., modifying a target nucleic acid) using the recited composition (claims 49 and 52).
Alignment of instant SEQ ID NO: 40 and SEQ ID NO: 922 of ‘575
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63
422
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However, ‘575 does not teach the Cas12i2 domain is fused to a heterologous sequence comprising i) a fusion domain with at least 88% identity to SEQ ID NO: 61 and ii) at least one linker sequence.
Cheng et al teach a Cas12i2 fusion protein comprising: (a) a Cas12i2 domain comprising an amino acid sequence of SEQ ID NO: 5, which is 99.6% identical to instant SEQ ID NO: 40, comprising amino acid substitutions of an arginine at positions 581 and 926, and a valine at position 1030 (pg. 60-61); and (b) a heterologous sequence comprising a fusion domain comprising an amino acid sequence of SEQ ID NO: 301, which is 100% identical to instant SEQ ID NO: 61 (i.e., KRPAATKKAGQAKKKK). Cheng et al further teach at least one NLS is attached to the N-terminal and/or C-terminal of the Cas12i2 domain for “optimal expression and nuclear targeting in eukaryotic cells, e.g., human cells” ([0219]). Cheng et al also teach a variety of linkers that connect the Cas12i2 domain to an appended domain, e.g., localization factors, and these linkers include, but are not limited to, flexible glycine-serine linkers in various combinations and lengths, rigid linkers, as well as different combinations thereof listed in Table 11 ([0405]-[0406]).
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 Cas12i2 domain of ‘575 by fusing to at least one NLS of SEQ ID NO: 61 via optimized linkers taught by Cheng et al because it would have merely amounted to a simple combination of prior art elements according to known methods to yield predictable results. Each element in the combination (Cas12i2 domain and NLS) merely performs the same function as it does separately (Cas12i2 domain interacts with an RNA guide to perform gene editing and NLS directs entry of the Cas12i2 domain into the nucleus). One would have been motivated to have done so for the advantage of optimizing expression of Cas12i2 fusion protein and nuclear targeting in eukaryotic cells taught by Cheng et al. One would have had a reasonable expectation of success in doing so because Cheng et al already teach a Cas12i2 fusion protein comprising the NLS of SEQ ID NO: 61 and a Cas12i2 domain that is only one amino acid difference to SEQ ID NO: 40.
This is a provisional nonstatutory double patenting rejection.
Claims 50-52, 54-67 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 23, 28, 36-37, 41-42, and 47 of copending Application No. 18/000,218 in view of Chong et al (WO 2021/202800 A1; Published Date: Oct 07, 2021) and Cheng et al (US 2020/0063126 A1; Published Date: Feb 27, 2020).
Regarding instant claims 50-52, 54-67, ‘218 teaches a Cas12i2 fusion protein comprising: (a) a Cas12i2 domain of SEQ ID NO: 2, which is 99.6% identical to instant SEQ ID NO: 40, comprising an arginine at positions 581 and 926 but a valine at position 1030; and (b) a heterologous sequence comprising of at least one nuclear localization signal (NLS) (claim 23). ‘218 further teach a system or composition comprising nucleic acids encoding for the Cas12i2 domain and an RNA guide (i.e., first and second nucleic acid), wherein the RNA comprises a direct repeat sequence and a spacer sequence that binds to a target nucleic acid, and wherein the Cas12i2 domain binds to the RNA guide (claim 1). ‘218 further teach a vector comprising the nucleic acid of the Cas12i2 fusion protein (claims 28 and 36), and a cell comprising the Cas12i2 fusion protein (claims 37 and 47). ‘218 further teach a method of targeting a sequence a cell (i.e., modifying a target nucleic acid) using the recited composition (claims 41-42).
Alignment of instant SEQ ID NO: 40 and SEQ ID NO: 2 of ‘218
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68
427
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Greyscale
However, ‘218 does not teach wherein the Cas12i2 domain comprises a glycine at position 1030.
Chong et al teach a Cas12i2 fusion protein comprising: (a) a Cas12i2 domain comprising an amino acid sequence of SEQ ID NO: 4, which is 100% identical to instant SEQ ID NO: 40, wherein the Cas12i2 domain comprises an arginine at positions 581 and 926, and a glycine at position 1030; and (b) a heterologous sequence comprising a fusion domain comprising at least one nuclear localization signal (NLS) (pg. 69, para. 2). Chong et al further teach mutations in SEQ ID NO: 4 exhibits increased stability and/or protein-DNA interactions, specifically V1030G is at the C-terminal portion of the Cas12i2 domain (FIG. 20B, pg. 34, lines 1-3).
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 Cas12i2 domain of ‘218 to comprise mutation V1030G taught by Chong et al because it would have merely amounted to a simple substitution of prior art elements according to known methods to yield predictable results. One would have been motivated to have done so for the advantage of obtaining a Cas12i2 variant with increased stability and capable of forming binary complexes with decreased dissociations with RNA guides, as well as modifying a target nucleic acid using this Cas12i2 variant of Chong et al. One would have had a reasonable expectation of success in doing so because Chong et al already teach a Cas12i2 domain that is only one amino acid difference to SEQ ID NO: 2 of ‘218.
However, ‘218 does not teach the Cas12i2 domain is fused to a heterologous sequence comprising i) a fusion domain with at least 88% identity to SEQ ID NO: 61 and ii) at least one linker sequence.
Cheng et al teach a Cas12i2 fusion protein comprising: (a) a Cas12i2 domain comprising an amino acid sequence of SEQ ID NO: 5, which is 99.6% identical to instant SEQ ID NO: 40, comprising amino acid substitutions of an arginine at positions 581 and 926, and a valine at position 1030 (pg. 60-61); and (b) a heterologous sequence comprising a fusion domain comprising an amino acid sequence of SEQ ID NO: 301, which is 100% identical to instant SEQ ID NO: 61 (i.e., KRPAATKKAGQAKKKK). Cheng et al further teach at least one NLS is attached to the N-terminal and/or C-terminal of the Cas12i2 domain for “optimal expression and nuclear targeting in eukaryotic cells, e.g., human cells” ([0219]). Cheng et al also teach a variety of linkers that connect the Cas12i2 domain to an appended domain, e.g., localization factors, and these linkers include, but are not limited to, flexible glycine-serine linkers in various combinations and lengths, rigid linkers, as well as different combinations thereof listed in Table 11 ([0405]-[0406]).
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 Cas12i2 domain of ‘218 by fusing to at least one NLS of SEQ ID NO: 61 via optimized linkers taught by Cheng et al because it would have merely amounted to a simple combination of prior art elements according to known methods to yield predictable results. Each element in the combination (Cas12i2 domain and NLS) merely performs the same function as it does separately (Cas12i2 domain interacts with an RNA guide to perform gene editing and NLS directs entry of the Cas12i2 domain into the nucleus). One would have been motivated to have done so for the advantage of optimizing expression of Cas12i2 fusion protein and nuclear targeting in eukaryotic cells taught by Cheng et al. One would have had a reasonable expectation of success in doing so because Cheng et al already teach a Cas12i2 fusion protein comprising the NLS of SEQ ID NO: 61 and a Cas12i2 domain that is only one amino acid difference to SEQ ID NO: 40.
This is a provisional nonstatutory double patenting rejection.
Claims 50-52, 54-67 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 3, 12, 15, and 28 of copending Application No. 18/442,529 in view of Cheng et al (US 2020/0063126 A1; Published Date: Feb 27, 2020).
Regarding instant claims 50-52, 54-67, ‘529 teaches a Type V CRISPR nuclease (i.e., Cas12i2 domain) of SEQ ID NO: 3, which is 100% identical to instant SEQ ID NO: 40, comprising an arginine at positions 581 and 926 and a glycine at position 1030 (claim 3). ‘529 further teach a system or composition comprising vectors or nucleic acids encoding for the Cas12i2 domain and an RNA guide (i.e., first and second nucleic acid), wherein the RNA comprises a direct repeat sequence and a spacer sequence that binds to a target nucleic acid, and wherein the Cas12i2 domain binds to the RNA guide (claim 12). ‘529 further teach a method of inhibiting aberrant splicing in a Stathmin-2 transcript a cell (i.e., modifying a target nucleic acid) using the recited composition (claims 15 and 28).
However, ‘529 does not teach the Cas12i2 domain is fused to a heterologous sequence comprising i) a fusion domain with at least 88% identity to SEQ ID NO: 61 and ii) at least one linker sequence.
Cheng et al teach a Cas12i2 fusion protein comprising: (a) a Cas12i2 domain comprising an amino acid sequence of SEQ ID NO: 5, which is 99.6% identical to instant SEQ ID NO: 40, comprising amino acid substitutions of an arginine at positions 581 and 926, and a valine at position 1030 (pg. 60-61); and (b) a heterologous sequence comprising a fusion domain comprising an amino acid sequence of SEQ ID NO: 301, which is 100% identical to instant SEQ ID NO: 61 (i.e., KRPAATKKAGQAKKKK). Cheng et al further teach at least one NLS is attached to the N-terminal and/or C-terminal of the Cas12i2 domain for “optimal expression and nuclear targeting in eukaryotic cells, e.g., human cells” ([0219]). Cheng et al also teach a variety of linkers that connect the Cas12i2 domain to an appended domain, e.g., localization factors, and these linkers include, but are not limited to, flexible glycine-serine linkers in various combinations and lengths, rigid linkers, as well as different combinations thereof listed in Table 11 ([0405]-[0406]).
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 Cas12i2 domain of ‘529 by fusing to at least one NLS of SEQ ID NO: 61 via optimized linkers taught by Cheng et al because it would have merely amounted to a simple combination of prior art elements according to known methods to yield predictable results. Each element in the combination (Cas12i2 domain and NLS) merely performs the same function as it does separately (Cas12i2 domain interacts with an RNA guide to perform gene editing and NLS directs entry of the Cas12i2 domain into the nucleus). One would have been motivated to have done so for the advantage of optimizing expression of Cas12i2 fusion protein and nuclear targeting in eukaryotic cells taught by Cheng et al. One would have had a reasonable expectation of success in doing so because Cheng et al already teach a Cas12i2 fusion protein comprising the NLS of SEQ ID NO: 61 and a Cas12i2 domain that is only one amino acid difference to SEQ ID NO: 40.
This is a provisional nonstatutory double patenting rejection.
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