DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 02/24/2026 has been entered.
Claims 1 and 37 were amended in the claim set filed 01/14/2026. It is noted that the amendments to the claims filed on 01/14/2026 do not comply with the requirements of 37 CFR 1.121(c) because not all deletions are properly denoted. For example, at amended instant claim 37, the term “large nucleic acid” was amended to “nucleic acid” without properly striking through or otherwise marking “large” for deletion. However, in the interest of compact prosecution, the amendments to the claims have been entered.
Claims 33 and 47 were cancelled in the claim set filed 01/14/2026.
Accordingly, claims 1, 7, 8, 11, 15, 16, 20, 24, 26, 32, 36, and 37 are pending.
Status of Prior Rejections
RE: Claim Rejections - 35 USC § 102
►Claim 37 was previously rejected under 35 U.S.C. 102(a)(1) and 102(a)(2) as being anticipated by WO 2020/525361 A1 (hereinafter Diamant; as cited in the IDS filed 09/19/2023; of record).
Applicant has traversed the rejection of record, asserting that amended claim 37 is not anticipated by Diamant.
In response, this is found persuasive. However, new grounds of rejection necessitated by amendment are set forth below.
►Claim 47 was previously rejected under 35 U.S.C. 102(a)(1) and 35 U.S.C. 102(a)(2) as being anticipated by US 2020/0109398 (hereinafter Rubens; of record).
The cancellation of claim 47 renders the rejection thereof moot.
RE: Claim Rejections - 35 USC § 103
►Claims 1, 7, 8, 11, 15, 16, 20, 24, 26, and 36 are rejected under 35 U.S.C. 103 as being unpatentable over US 2020/0109398 (hereinafter Rubens; of record) in view of WO 2020/525361 A1 (hereinafter Diamant; as cited in the IDS filed 09/19/2023; of record) and Shen et al., 2014 (hereinafter Shen; of record).
Applicant has traversed the rejection of record, asserting that amended claim 1 is not obvious over the cited art. Applicant further asserts that the rationale for combining references is inadequate, as the art does not articulate any reason to substitute components from one gene editing system for another or any advantages that would flow from doing so.
In response, the arguments regarding the previously cited art not rendering amended claim 1 obvious is found persuasive. However, new grounds of rejection necessitated by amendment are set forth below.
Furthermore, in response to applicant’s argument that there is no teaching, suggestion, or motivation to combine the references, the examiner recognizes that obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007). In this case, while Anzalone does not explicitly teach that the pegRNA system disclosed therein may be used in other gene editing applications, it is common practice in the field to adapt systems or technologies developed for one purpose to improve new and existing systems or technologies. As reviewed in Anzalone et al., 2020 (hereinafter Anzalone), prime editing belongs to one class of CRISPR-based genome editing agents, including nucleases, base editors, transposases/recombinases, and prime editors (Figure 1). As depicted in Figure 1 of Anzalone, prime editors are capable of introducing PAM-proximal point mutations, small insertions, and small deletions; whereas, Cas transposes/recombinases are capable of introducing large insertions. While these Cas transposes/recombinases disclosed in Anzalone do not specifically read on the instantly claimed Cas-R2 retrotransposon fusions, they are broadly drawn to Cas and dCas fusions to transposons for CRISPR-associated transposon-mediated genomic integration (page 836, column 1, paragraphs 2-4; page 836, column 2, paragraph 3-page 837, column 1, paragraph 1; Figure 6). The disclosure of Anzalone thus establishes that prime editing and CRISPR-associated transposon-mediated gene editing were both known in the field and were known to accomplish different editing outcomes prior to the effective filing date of the instant invention. Therefore, based on the knowledge generally available to one of ordinary skill in the art prior to the effective filing date of the instant invention, it is considered that it would have been obvious to one of ordinary skill in the art to experiment with combining these various genome editing technologies for purposes of optimizing the desired editing outcome, as different technologies accomplish different types of editing events (even simultaneously), as depicted in Figure 1 of Anzalone. Therefore, all the principles of the claimed system were known prior to the effective filing date of the instant application.
►Claim 32 was previously rejected under 35 U.S.C. 103 as being unpatentable over US 2020/0109398 (hereinafter Rubens; of record) in view of WO 2020/525361 A1 (hereinafter Diamant; as cited in the IDS filed 09/19/2023; of record) and Shen et al., 2014 (hereinafter Shen; of record) as applied to claim 1, and further in view of Glass et al., 2018 (hereinafter Glass; of record) and WO 2021/046243 A2 (hereinafter Getts; as cited in the IDS filed 12/02/2024; of record), as evidenced by Palazzo and Lee, 2018 (hereinafter Palazzo; of record).
Applicant has traversed the rejection of record, asserting that the number of cited references is “enormous” and reflects “cherry-pick[ing]” of certain sentences in each of the references to combine in hindsight view of pending claim 32.
In response, while Applicant’s argument has been fully considered, this is not found persuasive. However, for purposes of improving the clarity of the rejection of record, new art has been applied to explicitly motivate the claimed nuclear retention element as well as to address the amended claim limitations.
In response to applicant’s argument that the examiner has combined an excessive number of references, reliance on a large number of references in a rejection does not, without more, weigh against the obviousness of the claimed invention. See In re Gorman, 933 F.2d 982, 18 USPQ2d 1885 (Fed. Cir. 1991).
Furthermore, in response to applicant’s argument that the examiner’s conclusion of obviousness is based upon improper hindsight reasoning, it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant’s disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971).
The prior art supports this rationale. As set forth in the rejection of record, Glass discloses that CRISPR machinery must be directly transported to the nucleus to access the genomic DNA and to function effectively, while Getts and Palazzo both disclose that nuclear retention signal sequences (or elements) are useful to increase nuclear retention. The rejection of record therefore reasoned that one of ordinary skill in the art would have been motivated to engineer the payload RNA carrying a nucleic acid sequence for insertion to further comprise a nuclear retention element such that it is capable of being retained in the nucleus to facilitate said insertion. In support of this rationale, Ma et al., 2016 (hereinafter Ma) discloses that nuclear Cas9-guide RNA complex level limit targeting efficiency (abstract), with nuclear guide RNA concentration specifically being a limiting factor for efficient and effective DNA targeting (page 530, column 2, paragraph 3; Figure 4). While Ma is generally drawn to dwell time as a result of gRNA mismatches or the effect of gRNA truncation, Ma nonetheless clearly establishes that nuclear levels of CRISPR machinery, specifically the RNAs associated with CRISPR machinery for purposes of genome targeting, are crucial to successful CRISPR-based genome targeting and subsequent editing, as in the instant system. The Examiner notes that Ma does not specifically disclose that nuclear retention (including nuclear retention accomplished with a nuclear retention element) is crucial for payload RNA, as instantly claimed. However, as set forth above regarding the pegRNAs of Anzalone, it is common practice in the field to adapt systems or technologies developed for one purpose to improve new and existing systems or technologies.
Furthermore, as set forth in the previous action, fusion of nuclear retention signal sequences to retrotransposons is well-known in the art, as Getts discloses such fusion to LINE-1 retrotransposons for purposes of genome editing (paragraph [00238]). As is known to those of ordinary skill in the art and disclosed in Getts, LINE-1 retrotransposons are non-LTR retrotransposons (paragraph [0023]), as are the R2 elements of the instant application. As previously set forth, there is nothing in the art to suggest that nuclear retention signal sequences are only compatible with LINE-1 retrotransposons, as both R2 elements and LINE-1 retrotransposons are non-LTR retrotransposons (as set forth above) and nuclear retention signal sequence (or elements) have been shown to be functionally compatible with diverse biological species, including retrotransposons (as set forth above) and even mRNA (as disclosed in Palazzo: see section “Reporter mRNAs”-pages 2-3). Therefore, all the principles of the claimed system were known prior to the effective filing date of the instant application.
New/Maintained Grounds of Rejection
Claim Interpretation
With regard to claim 16, which directly depends from amended instant claim 1 and recites “the genome editing system of claim 1, wherein the Cas12 protein is fully active or catalytically dead,” the Examiner notes that independent claim 1 recites fusion of “the R2 element enzyme…to a Cas9 or Cas12 protein having nickase activity.” Thus, the genome editing system of claim 1 requires either a Cas9 protein having nickase activity or a Cas12 protein having nickase activity. The recitation of dependent claim 16 does not require fusion to a Cas12 protein. Therefore, any genome editing system comprising an R2 element enzyme fused to a Cas9 nickase and satisfying all other limitations of independent claim 1 must also read on the genome editing system of dependent claim 16, as Cas12 fusion is not required by the instant claim language.
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 1, 7, 8, 11, 15, 16, 20, 24, 26, 32, 36, and 37 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Claims 1 and 37 are drawn to a genome editing system and a method comprising the same, wherein said system and method comprise a Cas9 or a Cas12 protein having nickase activity. Thus, the claims are drawn to a genus of Cas9 or Cas12 proteins having nickase activity. Dependent claims 7, 8, 11, 15, 16, 20, 24, 26, 32, and 36 all directly depend from claim 1. While dependent claim 15 further limits the claims Cas9 nickase to comprise specific mutations, these mutations include catalytically dead Cas9 species (H840A/D10A), which is not compatible with nickase activity, as set forth below.
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 Cas9 H840A nickase variants (see Examples 10-13 and 15). No description is provided of Cas12 nickase variants beyond broad disclosure of a Cas12 protein functioning as a nickase, with no associated mutations specifically indicated (see paragraphs [0012], [0030], [0148]).
Furthermore, while the instant specification discloses that H840A/D10A Cas9 variants are catalytically dead Cas9 variants (paragraphs [0012] and [0197]), the instant claim set is drawn to Cas9 nickase species (as recited at independent instant claim 1). No description or working example is provided of a Cas9 nickase variant comprising the mutations H840A/D10A, as is instantly claimed.
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 described therein are only representative of Cas9 H840A nickase variants. The results are not necessarily predictive of any Cas9 or Cas12 nickase variant, nor are they necessarily predictive of the activity of a catalytically dead Cas9 variant such as one comprising the mutations H840A/D10A, as set forth above. Thus, it is impossible for one to extrapolate from the few examples described herein those Cas9 or Cas12 nickase variants 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. Regarding the instantly claimed Cas9 nickase variants, the prior art does not disclose a Cas9 nickase variant comprising the mutations H840A/D10A, as is instantly claimed. Furthermore, while Cas9 nickase variants D10A and H840A are known in the art, they are mechanistically distinct in that D10A is mutated in the RuvC domain; whereas, H840A is mutated in the HNH domain (Gopalappa et al., 2018: page 2, column 1, paragraph 2). It is also known that Cas9 RuvC and HNH domains have different cleavage activities, with the Cas9 HNH domain having higher cleavage activity than the RuvC domain (Gopalappa et al., 2018: section “The Cas9 HNH domain has higher cleavage activity than the RuvC domain). Additionally, HNH-mutant Cas9 species produce 3’ overhangs primarily engaged in insertions featuring microhomologies; whereas, RuvC-mutant Cas9 species produce 5’ overhangs that generate high levels of gene correction by homology-directed repair (Gopalappa et al., 2018: page 8, column 1, paragraph 2; page 8, column 2, paragraph 2). Thus, not all Cas9 nickase mutants function identically/equally, nor are all Cas9 nickase mutants well-suited for identical genome editing applications.
Additionally, while Cas9 nickase variants are known in the art (as set forth above), efforts to generate a catalytically efficient Cas12a nickase (which is a Cas12 species, as instantly claimed) have proved challenging, as Cas12a utilizes only a single catalytic site for cleavage of both DNA strands (Swarts and Jinek, 2018 (of record): page 13, paragraph 4). The instant application is silent as to the design of a Cas12 nickase such as a Cas12a nickase.
Furthermore, with specific regard to dependent claim 7, which recites “the R2 element enzyme is modified by an N-terminal truncation, an N-terminal insertion, or a modification to a -1 domain, 0 domain, or zinc finger domain,” the recited modifications/mutations are not limited in scope. Therefore, they necessarily encompass truncation or insertion of any number of residues from the N-terminus, as well as truncation, insertion, or modification of any number of residues from the -1 domain, 0 domain, or zinc finger domain. While the instant specification discloses truncation of the N terminus and removal or modification of the -1 domain, 0 domain, and zinc finger domains (Example 4), these working examples themselves disclose that there is a limit to the amount of N-terminal truncation that a single R2 element enzyme can tolerate while maintaining integration efficiency (paragraph [0179]) and that deletion of the -1 domain and/or 0 domain(s) significantly impairs integration efficiency (paragraph [0182]). The instant claim set necessarily encompasses such non-functional truncations/deletions but does not recite any further limitations that would clarify how such R2 species would be reasonably expected to function as instantly claimed.
Therefore, the skilled artisan would have reasonably concluded applicants were not in possession of the claimed invention for claims 1, 7, 8, 11, 15, 16, 20, 24, 26, 32, 36, and 37.
Claim Rejections - 35 USC § 112(d)
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.
Claim 16 is 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.
Claim 16, which recites that “the Cas12 protein is fully active or catalytically dead,” directly depends from instant claim 1, which recites “a…Cas12 protein having nickase activity.” Thus, the Cas12 protein with nickase activity of independent claim 1 is substituted with a Cas12 protein that is fully active or catalytically dead. As is known to those of ordinary skill in the art, CRISPR-Cas enzymes (such as those from Type II and Type V systems) possess nucleolytic activity that generates double-stranded breaks of the targeted DNA strands (reviewed in Brezgin et al., 2019: see particularly page 1, paragraphs 1 and 2 of “1. Introduction”). However, these enzymes have been successfully mutated to form nickase variants that make single-stranded DNA cuts (or nicks) and catalytically dead variants devoid of nucleolytic activity (Brezgin et al., 2019: abstract; page 12, paragraph 2 of “6.1. DNA Editing Using dCas Tools”). These are all distinct enzyme variants with distinct functions. Therefore, it cannot be considered that a Cas12 protein having nickase activity, as required by independent claim 1, encompasses Cas12 proteins that are catalytically dead or fully active, as recited at dependent claim 16.
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 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 1, 7, 8, 11, 15, 16, 20, 24, 26, 32, 36, and 37 are rejected under 35 U.S.C. 103 as being unpatentable over WO 2021/178709 A1 (hereinafter Steinberg; as cited in the IDS filed 09/19/2023) in view of Anzalone et al., 2020 (hereinafter Anzalone) and WO 2020/525361 A1 (hereinafter Diamant; as cited in the IDS filed 09/19/2023; of record), as evidenced by Burke et al., 1999 (hereinafter Burke; of record) and Palazzo and Lee, 2018 (hereinafter Palazzo; of record).
With regard to amended claim 1, which recites "a genome editing system comprising:
i) an R2 element enzyme having an N-terminus and a C-terminus, wherein the R2 element enzyme comprises a reverse transcriptase domain, a nickase domain, and a nuclear localization signal (NLS) at the N-terminus or the C-terminus, wherein the R2 element enzyme is fused to a Cas9 or Cas12 protein having nickase activity;
ii) at least one guide RNA; and
iii) a payload RNA, wherein the payload RNA comprises a protein binding element and an insertion template comprising a nucleic acid sequence for insertion in a genome, and wherein the payload RNA further comprises a Cas9 or Cas12 guide RNA, wherein the Cas9 or Cas12 guide RNA comprises an extension with a 5’ homology sequence, a 3’ homology sequence, a 5’ untranslated region (UTR), a 3’ UTR, an insertion template, or any combination thereof," Steinberg discloses methods and compositions for modulating a genome (abstract), said methods and compositions including further modifications to the Gene Writer™ system (reviewed from page 89, line 5-page 91, line 8). The Gene Writer™ system is built on functional modularization of non-LTR retrotransposons to target, edit, modify, or manipulate a target DNA sequence (i.e. to insert a heterologous nucleic acid sequence) by reverse transcription (page 89, lines 28-31). As depicted in Figures 29A and 29B, the modular components of the Gene Writer™ system include a DNA binding domain, an RNA binding domain, a reverse transcriptase domain, and an endonuclease domain (Figure 29A). A wild-type retrotransposase Gene Writer™ may be an R2 element enzyme comprising all four modular domains (Figure 29B; page 90, lines 12-13); whereas, a Cas9 retargeted Gene Writer may comprise a DNA binding and/or endonuclease domain from a Cas9 enzyme such as a Cas9 nickase (Figures 29A and 29B). The Gene Writer™ system of Steinberg is further disclosed to comprise a nuclear localization signal fused to the N-terminus or C-terminus of a Gene Writer™ as described therein (i.e. encompassing a wild type R2 Gene Writer™) (Figure 29B; page 151, lines 17-24) and as instantly claimed.
Regarding the instantly claimed R2 element enzyme comprising a reverse transcriptase domain, a nickase domain, and a targeting domain, these are considered to be endogenous domains of R2 element enzymes. As reviewed in Burke, all R2 elements recognize and nick a specific target sequence (the 28S gene sequence) via an endonuclease domain that nicks said target sequence (page 505, column 1, paragraph 2; page 508, column 1, paragraph 1). Accordingly, the nicking activity and specific targeting of R2 element enzymes is considered to read on the instantly claimed nickase domain and targeting domain. This is supported by the instant specification, as it is explicitly disclosed that the targeting domain may be a natural targeting domain (paragraph [0010]). Furthermore, as shown in Figure 1 of Burke, all R2 element enzymes also comprise a reverse transcriptase domain. Thus, the claimed R2 element enzyme domains are all considered to be endogenous R2 domains present in any R2 element enzyme.
Additionally, the Gene Writer™ system is further disclosed to comprise a guide RNA as part of the template RNA (embodiment 448: page 69), as well as a guide RNA to target a specified genomic site (page 557, lines 3-12). The template RNA of the Gene Writer™ system is considered to read on the instantly claimed payload RNA, as the template RNA comprises a sequence that binds the Gene Writer™ polypeptide, a heterologous insert sequence (page 90, lines 5-6), and a guide RNA (embodiment 448: page 69) with a 3’ extension to include donor sequence for genome modification (Figure 8B; page 847, lines 11-13) (i.e. a pegRNA as disclosed at page 589, lines 14-15). Such pegRNAs are disclosed in Anzalone, which reviews CRISPR-based genome editing agents, including prime editing, nucleases, base editors, and transposases/recombinases (Figure 1). As depicted in Figure 1 of Anzalone, prime editors are capable of introducing PAM-proximal point mutations, small insertions, and small deletions; whereas, Cas transposes/recombinases are capable of introducing large insertions. While these Cas transposes/recombinases disclosed in Anzalone do not specifically read on the instantly claimed Cas-R2 retrotransposon fusions, they are broadly drawn to Cas and dCas fusions to transposons for CRISPR-associated transposon-mediated genomic integration (page 836, column 1, paragraphs 2-4; page 836, column 2, paragraph 3-page 837, column 1, paragraph 1; Figure 6). The disclosure of Anzalone thus establishes that prime editing and CRISPR-associated transposon-mediated gene editing were both known in the field and were known to accomplish different editing outcomes prior to the effective filing date of the instant invention. Therefore, based on the knowledge generally available to one of ordinary skill in the art prior to the effective filing date of the instant invention, it is considered that it would have been obvious to one of ordinary skill in the art to experiment with combining these various genome editing technologies for purposes of optimizing the desired editing outcome(s), as different technologies accomplish different types of editing events (even simultaneously), as depicted in Figure 1 of Anzalone.
Thus, Steinberg discloses a genome editing system comprising fusion of R2 element enzymes (which may have a nuclear localization signal fused to the N-terminus thereof) and a Cas enzyme such as a Cas9 nickase, as well as at least one guide RNA and a payload RNA comprising a protein binding element, an insertion template, and a Cas9 guide RNA, said guide RNA comprising an extension with an insertion template (reviewed in Anzalone), as instantly claimed.
While one of ordinary skill in the art may argue that Steinberg discloses fusion of R2 element enzyme domains to a Cas enzyme such as a Cas9 nickase rather than fusion of R2 element enzymes in their entirety, this deficiency is cured by Diamant. As previously set forth, Diamant discloses a gene editing composition (abstract) comprising at least one fusion protein, itself comprising a retrotransposon-encoded protein portion linked to a CRISPR nuclease portion (i.e. Cas9 (paragraph [0086])), as well as an RNA template molecule comprising an insert template sequence (between 10 and 10,000 nucleotides in length (paragraph [0047])) and a guide portion (abstract, paragraph [0014]). Diamant explicitly discloses that the fusion protein of the gene editing composition taught therein comprises a CRISPR nuclease (i.e. SpCas9) with wild-type nuclease activity, a catalytically inactive nuclease, or nickase activity linked to a retrotransposon-encoded protein portion derived from R2 (and which may encode the full native protein) (paragraphs [0009], [0010], [0043], and [0132]).
Thus, Diamant clearly discloses that the genome editing system of Steinberg may comprise a full-length R2 element enzyme fused to a Cas enzyme such as a Cas9 nickase and retain its genome editing function, as instantly claimed. Accordingly, it is considered that Steinberg, Anzalone, and Diamant collectively disclose and/or motivate each and every limitation of amended instant claim 1.
With regard to claim 7, which recites “the R2 element enzyme [of the genome editing system of claim 1] is modified by an N-terminal truncation, an N-terminal insertion, or a modification to a -1 domain, 0 domain, or zinc finger domain,” Steinberg further discloses that the R2Tg retrotransposase (derived from Taeniopygia guttata: page 84, line 21) taught therein may comprise mutated zinc finger motifs (page 85, lines 15-18; Example 30). These mutated zinc finger motifs read on the instantly claimed modification to a zinc finger domain. Accordingly, it is considered that Steinberg discloses each and every additional limitation of instant claim 7.
With regard to claim 8, which recites “the R2 element enzyme [of the genome editing system of claim 1] comprises a linker,” Steinberg discloses that choice of linker can alter expression of Cas-RT fusions when fusing a Cas enzyme to the reverse transcriptase domain of a retrotransposase (page 88, lines 10-20; Figure 39). This linker experimentation reads on the instantly claimed linker. Accordingly, it is considered that Steinberg discloses each and every additional limitation of instant claim 8.
With regard to claim 11, which recites “the genome editing system of claim 1…comprises a sequence and/or structure for targeting a genomic locus other than a 28s rRNA locus,” Steinberg discloses that the genome editing system taught therein and set forth above may be engineered to target a genomic safe harbor site (i.e. by guide RNAs as disclosed at page 557, lines 3-12), including sites such as AAVS1 on chromosome 19 (page 76, line 25-page 77, line 9; page 668, lines 4-8). Given that AAVS1 is not a 28s rRNA locus, it is considered that AAVS1 targeting by the guide RNAs disclosed therein reads on the instantly claimed genomic locus target. Accordingly, it is considered that Steinberg discloses each and every additional limitation of instant claim 11.
With regard to claim 15, which recites “the Cas9 protein [of the genome editing system of claim 1] comprises the mutations H840A/D10A, H840A, or D10A,” as set forth above, Steinberg discloses that the Cas enzymes taught therein may be Cas9 nickases (Figures 29A and 29B). Such nickase species include D10A Cas9 mutants (Figure 5; Figure 8A; Figure 9A; Figure 10C), as instantly claimed. Furthermore, H840A is also disclosed as a suitable nickase mutant variant at page 576, line 8). Accordingly, it is considered that Steinberg discloses each and every additional limitation of instant claim 15.
With regard to claim 16, which recites “the Cas12 protein [of the genome editing system of claim 1] is fully active, catalytically dead, or functions as a nickase,” as set forth above, Steinberg discloses that the genome editing system taught therein may comprise a Cas9 enzyme such as a Cas9 nickase (Figures 29A and 29B). Additionally, as set forth above (see section Claim Interpretation), claim 16 does not require fusion of the R2 element enzyme of claim 1 to a Cas12 protein that is fully active or catalytically dead. Instead, claim 16 limits the claimed Cas12 protein, which is not required by the instant claim language. Thus, the Cas9 nickase disclosed in Figures 29A and 29B of Steinberg satisfies the requirement of a Cas9 nickase in both instant claims 1 and 16, while Diamant discloses fusion of the same to an R2 element enzyme, as set forth above. Accordingly, it is considered that Steinberg discloses each and every additional limitation of instant claim 16.
With regard to claim 20, which recites “the payload RNA [of the genome editing system of claim 1] further comprises one or more of a 5’ homology region or a 3’ homology region,” the template RNA of the Gene Writer™ system taught in Steinberg (set forth above) is disclosed to comprise 5’ and 3’ homology domains (Figure 19; page 83, lines 24-27), as instantly claimed. Accordingly, it is considered that Steinberg discloses each and every additional limitation of instant claim 20.
With regard to claim 24, which recites “the genome editing system [of claim 1], wherein the genome editing system functions in post-mitotic cells,” Steinberg discloses that the Gene Writer™ system taught therein is suitable for editing of non-dividing and terminally differentiated cells (i.e. post-mitotic cells) that have endogenously low levels of homologous recombination, as Gene Writing functions independently of homologous recombination (Example 16). This disclosure reads on the instantly claimed functioning in post-mitotic cells. Accordingly, it is considered that Steinberg discloses each and every additional limitation of instant claim 24.
With regard to claim 26, which recites “the payload RNA [of the genome editing system of claim 1] further comprises one or more of a 5’ untranslated region (UTR), a 3’ UTR, or both a 5’ UTR and a 3’ UTR,” the template RNA of the Gene Writer™ system taught in Steinberg (set forth above) is disclosed to comprise either or both of 5’ and 3’ UTRs (Figure 19; page 83, lines 24-27), as instantly claimed. Accordingly, it is considered that Steinberg discloses each and every additional limitation of instant claim 26.
With regard to claim 32, which recites “the genome editing system of claim 1, wherein the payload RNA further comprises a nuclear retention element,” Steinberg further discloses that the template RNA taught therein (which reads on the instantly claimed payload RNA, as set forth above) may comprise a SIRLOIN or MALAT1 signal (page 150, line 11-page 151, line 16). While Steinberg discloses that these signals are nuclear localization signals, those of ordinary skill in the art are aware that SIRLOIN and MALAT1 are more specifically known as nuclear retention elements, as reviewed in Palazzo (page 9, column 1, paragraph 2; page 9, column 2, paragraph 2). Thus, while Steinberg discloses SIRLOIN and MALAT1 signals as nuclear localization signals, they nonetheless read on the instantly claimed nuclear retention element attached to the template/payload RNA of the genome editing system taught therein. Accordingly, it is considered that Steinberg, as evidenced by Palazzo, discloses each and every additional limitation of instant claim 32.
With regard to claim 36, which recites “the insertion template [of the genome editing system of claim 1] comprises a template for a reporter gene, a transcription factor gene, a transgene, an enzyme gene, or a therapeutic gene,” the Gene Writer™ gene editing system taught in Steinberg (as set forth above) is disclosed to be useful for delivering therapeutic transgenes such as alglucosidase alpha for treatment of Pompe disease and galsulfase for treatment of mucopolysaccharidoses, among others (page 720, lines 6-15). These therapeutic transgenes read on the instantly claimed insertion template comprising a therapeutic gene. Accordingly, it is considered that Steinberg discloses each and every additional limitation of instant claim 36.
With regard to amended claim 37, which recites “a method of inserting a nucleic acid into a genome within a cell using a Cas9 or Cas12 fusion protein having nickase activity, wherein the method comprises supplying a Cas9 or Cas12 fusion protein to a cell, wherein the Cas9 or Cas12 fusion protein is supplied with at least one guide RNA, and a payload RNA template comprising a nucleic acid sequence, wherein the payload RNA further comprises a Cas9 or Cas12 guide RNA, wherein the RNA template is reverse transcribed by the Cas9 or Cas12 fusion protein prior to being inserted into the genome of the cell; and wherein the nucleic acid sequence is inserted into the genome of the cell; and wherein the Cas9 or Cas12 guide RNA comprises an extension with a 5’ homology sequence, a 3’ homology sequence, a 5’ untranslated region (UTR), a 3’ UTR, an insertion template, or any combination thereof,” as set forth above, Steinberg discloses the Gene Writer™ system comprising fusion of R2 element enzymes (which may have a nuclear localization signal fused to the N-terminus thereof) and a Cas enzyme such as a Cas9 nickase, as well as at least one guide RNA and a payload RNA comprising a protein binding element, an insertion template, and a Cas9 guide RNA, said guide RNA comprising an extension with an insertion template (i.e. a pegRNA: page 589, lines 14-15; reviewed in Anzalone), as instantly claimed. Furthermore, Steinberg discloses that the system taught therein reverse transcribes the template RNA sequence for insertion into the host genome, thereby modifying the host genome (page 90, lines 17-19), as instantly claimed.
While one of ordinary skill in the art may argue that Steinberg discloses fusion of R2 element enzyme domains to a Cas enzyme such as a Cas9 nickase rather than fusion of R2 element enzymes in their entirety, this deficiency is cured by Diamant. As previously set forth, Diamant discloses a gene editing composition (abstract) comprising at least one fusion protein, itself comprising a retrotransposon-encoded protein portion linked to a CRISPR nuclease portion (i.e. Cas9 (paragraph [0086])), as well as an RNA template molecule comprising an insert template sequence (between 10 and 10,000 nucleotides in length (paragraph [0047])) and a guide portion (abstract, paragraph [0014]). Diamant explicitly discloses that the fusion protein of the gene editing composition taught therein comprises a CRISPR nuclease (i.e. SpCas9) with wild-type nuclease activity, a catalytically inactive nuclease, or nickase activity linked to a retrotransposon-encoded protein portion derived from R2 (and which may encode the full native protein) (paragraphs [0009], [0010], [0043], and [0132]).
Additionally, as set forth above, pegRNAs are disclosed in Anzalone, which reviews CRISPR-based genome editing agents, including prime editing, nucleases, base editors, and transposases/recombinases (Figure 1). As depicted in Figure 1 of Anzalone, prime editors are capable of introducing PAM-proximal point mutations, small insertions, and small deletions; whereas, Cas transposes/recombinases are capable of introducing large insertions. While these Cas transposes/recombinases disclosed in Anzalone do not specifically read on the instantly claimed Cas-R2 retrotransposon fusions, they are broadly drawn to Cas and dCas fusions to transposons for CRISPR-associated transposon-mediated genomic integration (page 836, column 1, paragraphs 2-4; page 836, column 2, paragraph 3-page 837, column 1, paragraph 1; Figure 6). The disclosure of Anzalone thus establishes that prime editing and CRISPR-associated transposon-mediated gene editing were both known in the field and were known to accomplish different editing outcomes prior to the effective filing date of the instant invention. Therefore, based on the knowledge generally available to one of ordinary skill in the art prior to the effective filing date of the instant invention, it is considered that it would have been obvious to one of ordinary skill in the art to experiment with combining these various genome editing technologies for purposes of optimizing the desired editing outcome(s), as different technologies accomplish different types of editing events (even simultaneously), as depicted in Figure 1 of Anzalone.
Accordingly, it is considered that Steinberg, Diamant, and Anzalone collectively disclose and/or motivate each and every limitation of amended instant claim 37.
Given that Steinberg discloses all of the components of the claimed fusion-based genome editing system (i.e. an R2 element enzyme or domains thereof, a Cas enzyme such as a Cas9 or Cas12 enzyme/nickase, and a template/payload RNA further comprising a Cas9 or Cas12 guide RNA with an insertion template), that Diamant discloses that such fusion-based genome editing systems are functional when fusing the entirety of an R2 enzyme to a Cas enzyme, and that Anzalone discloses that prime editing facilitated by pegRNAs is amenable to producing small insertions and small deletions, while CRISPR-associated transposon-mediated gene editing is amenable to introducing large insertions, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to fuse an R2 element enzyme to a Cas enzyme (as disclosed in Diamant and Steinberg) and supply an R2 template/payload RNA further comprising a Cas-compatible pegRNA (as disclosed in Steinberg and Anzalone) to predictably produce a genome editing system capable of simultaneously inserting or deleting small DNA sequences and generating large insertions efficiently and precisely. One would have been motivated to make such a modification in order to receive the expected benefit of generating a genome editing system capable of inserting DNA sequences with single-nucleotide precision while simultaneously generating large insertions.
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.
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Claims 1, 11, 15, 20, and 37 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 7, 13, 17, 52, 68, and 76 of copending Application No. 17/778,192 (corresponds to US 2023/0040216 A1; as cited in the IDS filed 12/02/2024; claims amended 01/27/2026) in view of WO 2020/525361 A1 (hereinafter Diamant; as cited in the IDS filed 09/19/2023; of record), WO 2021/178709 A1 (hereinafter Steinberg; as cited in the IDS filed 09/19/2023) and Anzalone et al., 2020 (hereinafter Anzalone), as evidenced by Burke et al., 1999 (hereinafter Burke; of record).
Copending application ‘192 is drawn to systems and methods for targeted gene modification, said systems comprising components of CRISPR systems and non-LTR retrotransposon elements (abstract), as in the instant application.
Copending claim 1 recites “an engineered or non-naturally occurring composition comprising:
a site-specific nuclease polypeptide…;
a non-LTR retrotransposon polypeptide connected to…the site-specific nuclease polypeptide…;
a guide molecule capable of forming a complex with the site-specific nuclease polypeptide and directing site-specific binding to a target sequence of a target polynucleotide; and
a polynucleotide encoding a retrotransposon RNA, wherein the retrotransposon RNA comprises or encodes a donor polynucleotide.”
The claimed site-specific nuclease polypeptide is further recited to be a nickase or otherwise lack endonuclease activity at copending claim 7, while the claimed non-LTR retrotransposon polypeptide is recited to be R2 at copending claim 13. The claimed non-LTR retrotransposon polypeptide is further recited to comprise a nuclear localization signal at copending claim 17. The donor polynucleotide is further recited to comprise a homology sequence at copending claim 52 and a binding element capable of binding to the non-LTR retrotransposon polypeptide at copending claim 76.
Finally, copending claim 68 recites “a method of inserting a donor polynucleotide sequence into a target polynucleotide comprising: introducing the engineered or non-naturally occurring composition of claim 1 to a cell or population of cells, wherein the complex of the site-specific nuclease polypeptide and the guide directs the non-LTR retrotransposon polypeptide to the target sequence, and wherein the non-LTR retrotransposon polypeptide inserts the donor polynucleotide encoded by the retrotransposon RNA at, or adjacent to, the target sequence.”
In comparison, as set forth above, instant claim 1 recites "a genome editing system comprising:
i) an R2 element enzyme having an N-terminus and a C-terminus, wherein the R2 element enzyme comprises a reverse transcriptase domain, a nickase domain, and a nuclear localization signal (NLS) at the N-terminus or the C-terminus, wherein the R2 element enzyme is fused to a Cas9 or Cas12 protein having nickase activity;
ii) at least one guide RNA; and
iii) a payload RNA, wherein the payload RNA comprises a protein binding element and an insertion template comprising a nucleic acid sequence for insertion in a genome, and wherein the payload RNA further comprises a Cas9 or Cas12 guide RNA, wherein the Cas9 or Cas12 guide RNA comprises an extension with a 5’ homology sequence, a 3’ homology sequence, a 5’ untranslated region (UTR), a 3’ UTR, an insertion template, or any combination thereof." Thus, both the copending and instant applications are drawn to gene editing compositions, said compositions comprising an R2 element with a nuclear localization signal, a site-specific nuclease that is a nickase, and a donor polynucleotide within a retrotransposon RNA (i.e. the instantly claimed payload RNA). Furthermore, both the copending and instant applications recite that the systems taught therein further comprise a guide molecule (i.e. a guide RNA). However, the copending application does not recite that this composition must be a fusion with a nuclear localization signal at the N-terminus or C-terminus of the R2 element enzyme, nor does it recite that the payload RNA further comprises a Cas9 or Cas12 guide RNA with the recited modifications.
These deficiencies are cured by and Diamant, Steinberg, and Anzalone (as evidenced by Burke) as set forth below.
Regarding the claimed R2 element enzyme fusion, Diamant discloses a gene editing composition (abstract) comprising at least one fusion protein, itself comprising a retrotransposon-encoded protein portion linked to a CRISPR nuclease portion (i.e. Cas9 (paragraph [0086])), as well as an RNA template molecule comprising an insert template sequence (between 10 and 10,000 nucleotides in length (paragraph [0047])) and a guide portion (abstract, paragraph [0014]). Diamant explicitly discloses that the fusion protein of the gene editing composition taught therein comprises a CRISPR nuclease (i.e. SpCas9) protein portion (with wild-type nuclease, catalytically inactive nuclease, or nickase) linked to a retrotransposon-encoded protein portion derived from R2 (and which may encode the full native protein) (paragraphs [0009], [001 0], [0043], and [0132]). Thus, the fusion protein of Diamant, in combination with the recitation of the copending application, is not patentably distinct from the claimed subject matter of the instant application.
Regarding the instantly claimed R2 element enzyme comprising a reverse transcriptase domain, a nickase domain, and a targeting domain, these are considered to be endogenous domains of R2 element enzymes. As reviewed in Burke, all R2 elements recognize and nick a specific target sequence (the 28S gene sequence) via an endonuclease domain that nicks said target sequence (page 505, column 1, paragraph 2; page 508, column 1, paragraph 1). Accordingly, the nicking activity and specific targeting of R2 element enzymes is considered to read on the instantly claimed nickase domain and targeting domain. This is supported by the instant specification, as it is explicitly disclosed that the targeting domain may be a natural targeting domain (paragraph [0010]). Furthermore, as shown in Figure 1 of Burke, all R2 element enzymes also comprise a reverse transcriptase domain. Thus, the claimed R2 element enzyme domains are all considered to be endogenous R2 domains present in any R2 element enzyme.
Regarding the nuclear localization signal at the N-terminus of the R2 element enzyme, the Gene Writer™ system of Steinberg is further disclosed to comprise a nuclear localization signal fused to the N-terminus or C-terminus of a Gene Writer™ as described therein (i.e. encompassing a wild type R2 Gene Writer™) (Figure 29B; page 151, lines 17-24) and as instantly claimed.
Regarding the claimed guide RNA, as set forth above, the Gene Writer™ system of Steinberg is further disclosed to comprise a guide RNA as part of the template RNA (embodiment 448: page 69). The template RNA of the Gene Writer™ system is considered to read on the instantly claimed payload RNA, as the template RNA comprises a sequence that binds the Gene Writer™ polypeptide, a heterologous insert sequence (page 90, lines 5-6), and a guide RNA (embodiment 448: page 69) with a 3’ extension to include donor sequence for genome modification (Figure 8B; page 847, lines 11-13) (i.e. a pegRNA as disclosed at page 589, lines 14-15). Such pegRNAs are disclosed in Anzalone, which reviews CRISPR-based genome editing agents, including prime editing, nucleases, base editors, and transposases/recombinases (Figure 1). As depicted in Figure 1 of Anzalone, prime editors are capable of introducing PAM-proximal point mutations, small insertions, and small deletions; whereas, Cas transposes/recombinases are capable of introducing large insertions. While these Cas transposes/recombinases disclosed in Anzalone do not specifically read on the instantly claimed Cas-R2 retrotransposon fusions, they are broadly drawn to Cas and dCas fusions to transposons for CRISPR-associated transposon-mediated genomic integration (page 836, column 1, paragraphs 2-4; page 836, column 2, paragraph 3-page 837, column 1, paragraph 1; Figure 6). The disclosure of Anzalone thus establishes that prime editing and CRISPR-associated transposon-mediated gene editing were both known in the field and were known to accomplish different editing outcomes prior to the effective filing date of the instant invention. Therefore, based on the knowledge generally available to one of ordinary skill in the art prior to the effective filing date of the instant invention, it is considered that it would have been obvious to one of ordinary skill in the art to experiment with combining these various genome editing technologies for purposes of optimizing the desired editing outcome(s), as different technologies accomplish different types of editing events (even simultaneously), as depicted in Figure 1 of Anzalone.
Furthermore, instant claim 20 recites “the payload RNA [of the genome editing system of claim 1] further comprises one or more of a 5’ homology region or a 3’ homology region.” This is not patentably distinct from the recitation of copending claim 52, which recites that the donor polynucleotide (capable of binding to the non-LTR retrotransposon polypeptide per copending claim 76) further comprises “a homology sequence,” as instantly claimed. As is known to those of ordinary skill in the art (and disclosed in Steinberg), homology sequences are typically placed at the 5’ and/or 3’ region (depicted in Figure 19 of Steinberg), as instantly claimed.
With regard to instant claim 11, which recites “the genome editing system [of claim 1] comprises a sequence and/or structure for targeting a genomic locus other than a 28S rRNA locus,” as set forth above, while the copending application recites targeting a target sequence, it is not specifically recited that such a target sequence is not a 28S rRNA locus. However, this deficiency is cured by Steinberg. As set forth above, Steinberg discloses that the genome editing system taught therein and set forth above may be engineered to target a genomic safe harbor site, including sites such as AAVS1 on chromosome 19 (page 76, line 25-page 77, line 9; page 668, lines 4-8). Given that AAVS1 is not a 28s rRNA locus, it is considered that AAVS1 targeting reads on the instantly claimed genomic locus target.
With regard to instant claim 15, which recites “the Cas9 protein [of the genome editing system of claim 1] comprises the mutations H840A/D10A, H840A, or D10A,” as set forth above, the Cas polypeptide of the copending application is further recited to be a nickase at copending claim 7. However, the precise identity of this nickase is not recited in the copending application. This deficiency is cured by Steinberg, which discloses that a D10A mutation in Cas9 renders the Cas9 a nickase capable of functioning in the genome editing system taught therein (Figure 5; Figure 8A; Figure 9A; Figure 10C), as instantly claimed. Furthermore, H840A is also disclosed as a suitable nickase mutant variant at page 576, line 8).
With regard to amended claim 37, which recites “a method of inserting a nucleic acid into a genome within a cell using a Cas9 or Cas12 fusion protein having nickase activity, wherein the method comprises supplying a Cas9 or Cas12 fusion protein to a cell, wherein the Cas9 or Cas12 fusion protein is supplied with at least one guide RNA, and a payload RNA template comprising a nucleic acid sequence, wherein the payload RNA further comprises a Cas9 or Cas12 guide RNA, wherein the RNA template is reverse transcribed by the Cas9 or Cas12 fusion protein prior to being inserted into the genome of the cell; and wherein the nucleic acid sequence is inserted into the genome of the cell; and wherein the Cas9 or Cas12 guide RNA comprises an extension with a 5’ homology sequence, a 3’ homology sequence, a 5’ untranslated region (UTR), a 3’ UTR, an insertion template, or any combination thereof,” while copending claim 68 recites a method of inserting a donor polynucleotide sequence into a target polynucleotide, some of the instantly claimed method steps (i.e. reverse transcription of the template) and the guide RNA modifications are not recited in the copending application. However, this deficiency is cured by Diamant, Steinberg, and Anzalone (as evidenced by Burke).
Regarding the claimed R2 element enzyme fusion, Diamant discloses a gene editing composition (abstract) comprising at least one fusion protein, itself comprising a retrotransposon-encoded protein portion linked to a CRISPR nuclease portion (i.e. Cas9 (paragraph [0086])), as well as an RNA template molecule comprising an insert template sequence (between 10 and 10,000 nucleotides in length (paragraph [0047])) and a guide portion (abstract, paragraph [0014]). Diamant explicitly discloses that the fusion protein of the gene editing composition taught therein comprises a CRISPR nuclease (i.e. SpCas9) protein portion (with wild-type nuclease, catalytically inactive nuclease, or nickase) linked to a retrotransposon-encoded protein portion derived from R2 (and which may encode the full native protein) (paragraphs [0009], [001 0], [0043], and [0132]). Thus, the fusion protein of Diamant, in combination with the recitation of the copending application, is not patentably distinct from the claimed subject matter of the instant application.
Regarding the instantly claimed R2 element enzyme comprising a reverse transcriptase domain, a nickase domain, and a targeting domain, these are considered to be endogenous domains of R2 element enzymes. As reviewed in Burke, all R2 elements recognize and nick a specific target sequence (the 28S gene sequence) via an endonuclease domain that nicks said target sequence (page 505, column 1, paragraph 2; page 508, column 1, paragraph 1). Accordingly, the nicking activity and specific targeting of R2 element enzymes is considered to read on the instantly claimed nickase domain and targeting domain. This is supported by the instant specification, as it is explicitly disclosed that the targeting domain may be a natural targeting domain (paragraph [0010]). Furthermore, as shown in Figure 1 of Burke, all R2 element enzymes also comprise a reverse transcriptase domain. Thus, the claimed R2 element enzyme domains are all considered to be endogenous R2 domains present in any R2 element enzyme.
Regarding the claimed guide RNA, as set forth above, the Gene Writer™ system of Steinberg is further disclosed to comprise a guide RNA as part of the template RNA (embodiment 448: page 69). The template RNA of the Gene Writer™ system is considered to read on the instantly claimed payload RNA, as the template RNA comprises a sequence that binds the Gene Writer™ polypeptide, a heterologous insert sequence (page 90, lines 5-6), and a guide RNA (embodiment 448: page 69) with a 3’ extension to include donor sequence for genome modification (Figure 8B; page 847, lines 11-13) (i.e. a pegRNA as disclosed at page 589, lines 14-15). Such pegRNAs are disclosed in Anzalone, which reviews CRISPR-based genome editing agents, including prime editing, nucleases, base editors, and transposases/recombinases (Figure 1). As depicted in Figure 1 of Anzalone, prime editors are capable of introducing PAM-proximal point mutations, small insertions, and small deletions; whereas, Cas transposes/recombinases are capable of introducing large insertions. While these Cas transposes/recombinases disclosed in Anzalone do not specifically read on the instantly claimed Cas-R2 retrotransposon fusions, they are broadly drawn to Cas and dCas fusions to transposons for CRISPR-associated transposon-mediated genomic integration (page 836, column 1, paragraphs 2-4; page 836, column 2, paragraph 3-page 837, column 1, paragraph 1; Figure 6). The disclosure of Anzalone thus establishes that prime editing and CRISPR-associated transposon-mediated gene editing were both known in the field and were known to accomplish different editing outcomes prior to the effective filing date of the instant invention. Therefore, based on the knowledge generally available to one of ordinary skill in the art prior to the effective filing date of the instant invention, it is considered that it would have been obvious to one of ordinary skill in the art to experiment with combining these various genome editing technologies for purposes of optimizing the desired editing outcome(s), as different technologies accomplish different types of editing events (even simultaneously), as depicted in Figure 1 of Anzalone.
Furthermore, as set forth above, Steinberg discloses the Gene Writer™ system comprising fusion of R2 element enzymes (which may have a nuclear localization signal fused to the N-terminus thereof) and a Cas enzyme such as a Cas9 nickase, as well as at least one guide RNA and a payload RNA comprising a protein binding element, an insertion template, and a Cas9 guide RNA, said guide RNA comprising an extension with an insertion template (i.e. a pegRNA: page 589, lines 14-15; reviewed in Anzalone), as instantly claimed. Furthermore, Steinberg discloses that the system taught therein reverse transcribes the template RNA sequence for insertion into the host genome, thereby modifying the host genome (page 90, lines 17-19), as instantly claimed.
Given that both the copending and instant applications recite gene editing compositions, said compositions comprising an R2 element with a nuclear localization signal, a site-specific nuclease that is a nickase, and a donor polynucleotide within a retrotransposon RNA (i.e. the instantly claimed payload RNA), as well as a guide molecule such as a guide RNA, and that Diamant discloses that such a composition may be a fusion while Steinberg and Anzalone (as evidenced by Burke) disclose that the guide RNA of such a gene editing system may be a part of the payload (or retrotransposon or template) RNA and may further comprise an insert template to facilitate small insertions or deletions in addition to the large insertions facilitated by R2-Cas fusion, 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 the copending application per the disclosures of Diamant, Steinberg, and Anzalone to predictably produce a genome editing system capable of simultaneously inserting or deleting small DNA sequences and generating large insertions efficiently and precisely. One would have been motivated to make such a modification in order to receive the expected benefit of generating a genome editing system capable of inserting DNA sequences with single-nucleotide precision while simultaneously generating large insertions. Accordingly, it is considered that the copending and instant subject matter is not patentably distinct.
This is a provisional nonstatutory double patenting rejection.
Claims 1, 7, 8, 11, 32, and 37 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 2, 5, 7, 8, 11, 12, 14, 16, 32, 33, 35, and 37 of copending Application No. 18/301,732 (corresponds to US 2024/0035008 A1; as cited in the IDS filed 12/02/2024; claims amended 02/19/2026) in view of WO 2021/178709 A1 (hereinafter Steinberg; as cited in the IDS filed 09/19/2023).
Copending claim 1 recites “a genome editing system comprising:
an R2 element enzyme; and
a payload RNA, wherein the payload RNA comprises an insertion template and optionally one or more of a 5’ homology region, a 3’ homology region, and a protein binding element, wherein the insertion template comprises a sequence for a nucleic acid insertion into the genome, and wherein the R2 element enzyme comprises a reverse transcriptase domain, and a nickase domain.”
The copending R2 element enzyme is further recited to comprise a targeting domain at copending claim 2, to be modified by an N-terminal truncation at copending claims 5 and 7, to comprise a linker at copending claim 8, to be a fusion protein fused to a Cas12 protein that is fully active, catalytically dead, or functioning as a nickase at copending claims 14 and 16, and to comprise a nuclear localization signal at copending claim 35.
The copending payload RNA is further recited to comprise a Cas9 or Cas12 guide RNA comprising “an extension with a 5’ homology sequence, a 3’ homology sequence, a 5’ untranslated region (UTR), a 3’ UTR, an insertion template, or any combination thereof,” at copending claim 33.
In comparison, as set forth above, instant claim 1 recites "a genome editing system comprising:
i) an R2 element enzyme having an N-terminus and a C-terminus, wherein the R2 element enzyme comprises a reverse transcriptase domain, a nickase domain, and a nuclear localization signal (NLS) at the N-terminus or the C-terminus, wherein the R2 element enzyme is fused to a Cas9 or Cas12 protein having nickase activity;
ii) at least one guide RNA; and
iii) a payload RNA, wherein the payload RNA comprises a protein binding element and an insertion template comprising a nucleic acid sequence for insertion in a genome, and wherein the payload RNA further comprises a Cas9 or Cas12 guide RNA, wherein the Cas9 or Cas12 guide RNA comprises an extension with a 5’ homology sequence, a 3’ homology sequence, a 5’ untranslated region (UTR), a 3’ UTR, an insertion template, or any combination thereof." Thus, both the copending and instant applications are drawn to gene editing compositions, said compositions comprising an R2 element with a nuclear localization signal (as recited at copending claim 35), a targeting domain (as recited at copending claim 2) and fused to a site-specific nuclease that is a nickase (i.e. Cas12 as recited at copending claims 14 and 16). Furthermore, both the copending and instant applications recite a payload RNA comprising modifications including an extension with a 5’ homology sequence, a 3’ homology sequence, a 5’ untranslated region (UTR), a 3’ UTR, an insertion template, or any combination thereof” (copending claim 33)
However, the copending application does not recite that the system claimed therein comprises a guide RNA, nor does it recite that the nuclear localization signal must be located at the N-terminus or C-terminus of the R2 element enzyme. These deficiencies are cured by Steinberg, as set forth below.
As set forth above, the Gene Writer™ system of Steinberg is further disclosed to comprise a guide RNA to target a specified genomic site (page 557, lines 3-12). Furthermore, regarding the nuclear localization signal at the N-terminus of the R2 element enzyme, the Gene Writer™ system of Steinberg is further disclosed to comprise a nuclear localization signal fused to the N-terminus or C-terminus of a Gene Writer™ as described therein (i.e. encompassing a wild type R2 Gene Writer™) (Figure 29B; page 151, lines 17-24) and as instantly claimed.
Thus, the copending application, in view of Steinberg, discloses each and every limitation of instant claim 1.
With regard to instant claim 7, which recites “the R2 element enzyme [of the genome editing system of claim 1] is modified by an N-terminal truncation, an N-terminal insertion, or a modification to a -1 domain, 0 domain, or zinc finger domain,” as set forth above, copending claim 7 recites that the R2 element enzyme may be modified by an N-terminal truncation, as instantly claimed.
With regard to instant claim 8, which recites “the R2 element enzyme [of the genome editing system of claim 1] comprises a linker,” as set forth above, copending claim 8 also recites that the R2 element enzyme comprises a linker, as instantly claimed.
With regard to instant claim 11, which recites “the genome editing system [of claim 1] comprises a sequence and/or structure for targeting a genomic locus other than a 28S rRNA locus,” copending claim 11 recites “the genome editing system [claimed therein] targets a genomic locus other than the 28srRNA locus,” by modifying “an N-terminal zinc finger domain of the R2 element enzyme…to target a genomic locus other than the 28S rRNA locus” as recited at copending claim 12. Thus, the recitation of the copending application reads on the recitation of instant claim 11.
With regard to instant claim 32, which recites “the payload RNA [of the genome editing system of claim 1] further comprises a nuclear retention element,” copending claim 32 also recites that “the payload RNA [of the genome editing system claimed therein] further comprises a nuclear retention element.” Thus, the recitation of the copending application reads on the recitation of instant claim 11.
Finally, with regard to instant claim 37, which recites “a method of inserting a nucleic acid into a genome within a cell using a Cas9 or Cas12 fusion protein having nickase activity, wherein the method comprises supplying a Cas9 or Cas12 fusion protein to a cell, wherein the Cas9 or Cas12 fusion protein is supplied with at least one guide RNA, and a payload RNA template comprising a nucleic acid sequence, wherein the payload RNA further comprises a Cas9 or Cas12 guide RNA, wherein the RNA template is reverse transcribed by the Cas9 or Cas12 fusion protein prior to being inserted into the genome of the cell; and wherein the nucleic acid sequence is inserted into the genome of the cell; and wherein the Cas9 or Cas12 guide RNA comprises an extension with a 5’ homology sequence, a 3’ homology sequence, a 5’ untranslated region (UTR), a 3’ UTR, an insertion template, or any combination thereof,” copending claim 37 also recites a method of inserting a nucleic acid into a genome, said method comprising “supplying a Cas9 or Cas12 fusion protein to a cell, wherein the Cas9 or Cas12 fusion protein is supplied with a payload RNA template, wherein the RNA template is reverse transcribed by the Cas9 or Cas12 fusion protein prior to being inserted into the genome of the cell; and wherein the large nucleic acid is inserted into the genome of the cell.” Thus, the copending application recites every limitation of the instantly claimed method with the exception of supplying at least one guide RNA and the modifications to the guide RNA associated with the payload RNA of the instant application. However, as set forth above, the Gene Writer™ system of Steinberg is further disclosed to comprise a guide RNA to target a specified genomic site (page 557, lines 3-12), while the copending application recites identical payload RNA-associated guide RNA modifications at copending claim 33.
Given that both the copending and instant applications recite gene editing compositions and methods, said compositions comprising R2 element enzymes (comprising a nuclear localization signal) with the instantly claimed domains fused to a Cas12 nickase (including via a linker) as well as a payload RNA comprising an insert template to facilitate small insertions or deletions in addition to the large insertions facilitated by R2-Cas fusion, and that Steinberg discloses the Gene Writer™ system, which comprises at least one guide RNA to target a specified genomic site, as well as a nuclear localization signal at the N-terminus of the R2 element enzyme, 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 the copending application per the disclosure of Steinberg to include an additional guide RNA to target the genome editing composition to a specified genomic site, as well as to attach the nuclear localization signal to the N-terminus of the R2 element enzyme to predictably produce a genome editing system capable of simultaneously inserting or deleting small DNA sequences and generating large insertions efficiently and precisely. One would have been motivated to make such a modification in order to receive the expected benefit of generating a genome editing system capable of inserting DNA sequences with single-nucleotide precision while simultaneously generating large insertions. Accordingly, it is considered that the copending and instant subject matter is not patentably distinct.
This is a provisional nonstatutory double patenting rejection.
Claims 1, 7, 15, 16, and 20 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-3, 7, 11, 13, 14, 19, and 21 of copending Application No. 18/276,471 (corresponds to US 2024/0132916 A1; claims amended 02/29/2024) in view of WO 2021/178709 A1 (hereinafter Steinberg; as cited in the IDS filed 09/19/2023) and Anzalone et al., 2020 (hereinafter Anzalone), as evidenced by Burke et al., 1999 (hereinafter Burke; of record).
Copending claim 1 recites “an engineered composition for non-native, targeted transposition of donor sequence into targeted nucleic acids, comprising:
a first site-specific nuclease configured to bind a target sequence in a target polynucleotide;
a first non-LTR retrotransposon polypeptide fused to or otherwise capable of forming a complex with the first site-specific nuclease; and
a donor construct comprising, a donor polynucleotide sequence for insertion into the target polynucleotide and comprising one or more elements capable of forming a complex with the non-LTR retrotransposon polypeptide.”
The claimed site-specific nuclease is further recited to be a Cas polypeptide such as a Type II or Type V Cas polypeptide or nuclease thereof at copending claims 2 and 3, with specific recitation that Cas9 nickases comprising D10A or H840A mutations are compatible with the system of the copending application at copending claim 14. The non-LTR retrotransposon is fused to said site-specific nuclease per copending claim 13.
Furthermore, the claimed non-LTR retrotransposon polypeptide is further recited to be modified to form a dimer, wherein one polypeptide of the dimer may comprise nickase activity or truncations to or modifications of a zinc finger region to the same at copending claim 7. Suitable non-LTR retrotransposon polypeptides are recited to include R2 at copending claim 11.
Finally, the claimed donor construct is recited to further comprise “a first homology region, a donor template for insertion into the target polynucleotide, a second homology region, and a binding element capable of complexing with the non-LTR retrotransposon polypeptide” at copending claims 19 and 21.
In comparison, as set forth above, instant claim 1 recites "a genome editing system comprising:
i) an R2 element enzyme having an N-terminus and a C-terminus, wherein the R2 element enzyme comprises a reverse transcriptase domain, a nickase domain, and a nuclear localization signal (NLS) at the N-terminus or the C-terminus, wherein the R2 element enzyme is fused to a Cas9 or Cas12 protein having nickase activity;
ii) at least one guide RNA; and
iii) a payload RNA, wherein the payload RNA comprises a protein binding element and an insertion template comprising a nucleic acid sequence for insertion in a genome, and wherein the payload RNA further comprises a Cas9 or Cas12 guide RNA, wherein the Cas9 or Cas12 guide RNA comprises an extension with a 5’ homology sequence, a 3’ homology sequence, a 5’ untranslated region (UTR), a 3’ UTR, an insertion template, or any combination thereof." Thus, both the copending and instant applications are drawn to gene editing compositions, said compositions comprising an R2 element (as recited at copending claim 11), and fused to a site-specific nuclease that is a nickase (i.e. Type II or Type V Cas polypeptides and nucleases thereof as recited at copending claims 2, 3, 13, and 14). Furthermore, both the copending and instant applications recite a payload or donor RNA comprising modifications including an extension with a first and second homology region (copending claims 19 and 21)
However, the copending application does not recite that the system claimed therein comprises a guide RNA and an additional guide RNA associated with the payload or donor RNA, nor does it recite the R2 element enzyme must comprise a nuclear localization signal at its N-terminus or C-terminus. These deficiencies are cured by Steinberg, Anzalone, and Burke, as set forth below.
Regarding the instantly claimed R2 element enzyme comprising a reverse transcriptase domain, a nickase domain, and a targeting domain, these are considered to be endogenous domains of R2 element enzymes. As reviewed in Burke, all R2 elements recognize and nick a specific target sequence (the 28S gene sequence) via an endonuclease domain that nicks said target sequence (page 505, column 1, paragraph 2; page 508, column 1, paragraph 1). Accordingly, the nicking activity and specific targeting of R2 element enzymes is considered to read on the instantly claimed nickase domain and targeting domain. This is supported by the instant specification, as it is explicitly disclosed that the targeting domain may be a natural targeting domain (paragraph [0010]). Furthermore, as shown in Figure 1 of Burke, all R2 element enzymes also comprise a reverse transcriptase domain. Thus, the claimed R2 element enzyme domains are all considered to be endogenous R2 domains present in any R2 element enzyme.
Furthermore, as set forth above, the Gene Writer™ system of Steinberg is further disclosed to comprise a guide RNA as part of the template RNA (embodiment 448: page 69), as well as a guide RNA to target a specified genomic site (page 557, lines 3-12). The template RNA of the Gene Writer™ system is considered to read on the instantly claimed payload RNA, as the template RNA comprises a sequence that binds the Gene Writer™ polypeptide, a heterologous insert sequence (page 90, lines 5-6), and a guide RNA (embodiment 448: page 69) with a 3’ extension to include donor sequence for genome modification (Figure 8B; page 847, lines 11-13) (i.e. a pegRNA as disclosed at page 589, lines 14-15). Additionally, regarding the nuclear localization signal at the N-terminus or C-terminus of the R2 element enzyme, the Gene Writer™ system of Steinberg is further disclosed to comprise a nuclear localization signal fused to the N-terminus or C-terminus of a Gene Writer™ as described therein (i.e. encompassing a wild type R2 Gene Writer™) (Figure 29B; page 151, lines 17-24) and as instantly claimed. Finally, Steinberg discloses that Type II (such as Cas9) and Type V (such as Cas12) CRISPR-Cas polypeptides are suitable for use in the Gene Writer™ system (page 575, line 2-page 576, line 16), as in the copending (claims 2 and 3) and instant applications (claim 16).
Per Anzalone, prime editors are capable of introducing PAM-proximal point mutations, small insertions, and small deletions; whereas, Cas transposes/recombinases are capable of introducing large insertions (Figure 1). While these Cas transposes/recombinases disclosed in Anzalone do not specifically read on the instantly claimed Cas-R2 retrotransposon fusions, they are broadly drawn to Cas and dCas fusions to transposons for CRISPR-associated transposon-mediated genomic integration (page 836, column 1, paragraphs 2-4; page 836, column 2, paragraph 3-page 837, column 1, paragraph 1; Figure 6). The disclosure of Anzalone thus establishes that prime editing and CRISPR-associated transposon-mediated gene editing were both known in the field and were known to accomplish different editing outcomes prior to the effective filing date of the instant invention. Therefore, based on the knowledge generally available to one of ordinary skill in the art prior to the effective filing date of the instant invention, it is considered that it would have been obvious to one of ordinary skill in the art to experiment with combining these various genome editing technologies for purposes of optimizing the desired editing outcome(s), as different technologies accomplish different types of editing events (even simultaneously), as depicted in Figure 1 of Anzalone.
With regard to instant claim 7, which recites “the R2 element enzyme [of the genome editing system of claim 1] is modified by…a modification to a…zinc finger domain,” as set forth above, copending claim 7 also recites modifications of a zinc finger region of the non-LTR retrotransposon claimed therein (i.e. an R2 element enzyme as recited at copending claim 11).
With regard to instant claim 15, which recites “the Cas9 protein comprises the mutations H840A/D10A, H840A, or D10A,” as set forth above, copending claim 14 recites that Cas9 nickases comprising D10A or H840A mutations are compatible with the system of the copending application, as instantly claimed.
With regard to instant claim 16, which recites “the Cas12 protein [of the genome editing system of claim 1] is fully active or catalytically dead,” as set forth above, the copending application and Steinberg both disclose that Type II (i.e. Cas9) and Type V (i.e. Cas12) CRISPR-Cas polypeptides are suitable for use in the genome editing systems taught therein. Furthermore, copending claims 2 and 3 recite that the Type V Cas polypeptide may be a nickase variant.
Finally, with regard to instant claim 20, which recites “the payload RNA [of the genome editing system of claim 1] further comprises one or more of a 5’ homology region or a 3’ homology region,” as set forth above, copending claims 19 and 21 recite that the donor construct claimed therein further comprises first and second homology regions, as instantly claimed.
Given that both the copending and instant applications recite gene editing compositions, said compositions comprising an R2 element with a site-specific nuclease (that may be a nickase), and a donor polynucleotide within a retrotransposon RNA (i.e. the instantly claimed payload RNA as further disclosed by Steinberg and set forth above), and that Steinberg (as evidenced by Burke) further discloses that the guide RNA of such a gene editing system may be a part of the payload (or retrotransposon or template) RNA and may further comprise an insert template to facilitate small insertions or deletions in addition to the large insertions facilitated by R2-Cas fusion, as well as that the R2 element may comprise a nuclear localization signal fused to the N-terminus or C-terminus, 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 the copending application per the disclosure of Steinberg (as evidenced by Burke) to predictably produce a genome editing system capable of simultaneously inserting or deleting small DNA sequences and generating large insertions efficiently and precisely, as taught by Anzalone. One would have been motivated to make such a modification in order to receive the expected benefit of generating a genome editing system capable of inserting DNA sequences with single-nucleotide precision while simultaneously generating large insertions. Accordingly, it is considered that the copending and instant subject matter is not patentably distinct.
This is a provisional nonstatutory double patenting rejection.
Claims 1, 8, 16, 20, and 37 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 2, 13, 20, 21, 24, 32, and 44 of copending Application No. 19/333,523 (corresponds to US 2026/0008827 A1; claims amended 12/02/2025) in view of WO 2021/178709 A1 (hereinafter Steinberg; as cited in the IDS filed 09/19/2023) and Anzalone et al., 2020 (hereinafter Anzalone), as evidenced by Burke et al., 1999 (hereinafter Burke; of record).
Copending claim 1 recites “an engineered or non-naturally occurring composition for targeted transposition of a donor polynucleotide into a target polynucleotide, said composition comprising:
a programmable DNA-binding protein configured to bind a target sequence within a target polynucleotide;
a non-long terminal repeat (non-LTR) retrotransposon polypeptide fused to or otherwise capable of associating with the programmable DNA-binding protein, wherein the non-LTR retrotransposon polypeptide comprises one or more modifications or truncations relative to a wild-type non-LTR retrotransposon polypeptide; and
a donor construct comprising a donor polynucleotide for insertion into the target polynucleotide and an engineered binding element capable of forming a complex with the non-LTR retrotransposon polypeptide.”
The programmable DNA-binding protein of copending claim 1 is further recited to be “a CRISPR-Cas system comprising a Cas protein and one or more guide molecules capable of forming a complex with the Cas protein and directing sequence-specific binding of the complex to the target sequence within the target polynucleotide, optionally, wherein the CRISPR-Cas system is a Type II or Type V CRISPR-Cas system, or a Cas12i1 or Cas12i2 system, and optionally, wherein the Cas protein is a nickase” at copending claim 2.
The claimed non-LTR retrotransposon is further recited to be an “R2 polypeptide” fused to the programmable DNA-binding protein “by means of a flexible linker” at copending claims 13, 20, and 21.
The claimed donor construct is further recited to comprise “a first homology region, a donor template for insertion into the target polynucleotide, and a second homology region” at copending claim 24. Furthermore, the engineered binding element of the claimed donor construct (of copending claim 2) is further recited to be “fused to a 3’ or 5’ end of the one or more guide molecules” at copending claim 32.
Finally, the copending application recites “a method of inserting a donor polynucleotide into a target polynucleotide comprising introducing the composition of claim 1 into a cell or population of cells, wherein the programmable DNA-binding protein directs the non-LTR retrotransposon polypeptide to the target polynucleotide, and the non-LTR retrotransposon polypeptide inserts the donor polynucleotide into the target polynucleotide at or adjacent to the target sequence” at copending claim 44.
In comparison, as set forth above, instant claim 1 recites "a genome editing system comprising:
i) an R2 element enzyme having an N-terminus and a C-terminus, wherein the R2 element enzyme comprises a reverse transcriptase domain, a nickase domain, and a nuclear localization signal (NLS) at the N-terminus or the C-terminus, wherein the R2 element enzyme is fused to a Cas9 or Cas12 protein having nickase activity;
ii) at least one guide RNA; and
iii) a payload RNA, wherein the payload RNA comprises a protein binding element and an insertion template comprising a nucleic acid sequence for insertion in a genome, and wherein the payload RNA further comprises a Cas9 or Cas12 guide RNA, wherein the Cas9 or Cas12 guide RNA comprises an extension with a 5’ homology sequence, a 3’ homology sequence, a 5’ untranslated region (UTR), a 3’ UTR, an insertion template, or any combination thereof." Thus, both the copending and instant applications are drawn to gene editing compositions, said compositions comprising a non-LTR retrotransposon polypeptide such as an R2 element enzyme (as recited at copending claims 1, 13, and 20) fused to a Cas enzyme such as Cas12 with nickase activity (as recited at copending claims 2 and 21), at least one guide RNA (as recited at copending claim 2), and a donor construct or payload RNA comprising a protein binding element and a guide RNA compatible with CRISPR systems such as Cas12 systems (copending claims 2 and 32). Regarding the instantly claimed fusion protein, as set forth above, copending claim 21 recites fusion of the claimed non-LTR retrotransposon (i.e. R2) polypeptide to the programmable DNA-binding protein (i.e. Cas12) via a linker, as recited at instant claim 8.
However, the copending application does not recite the R2 element enzyme domains, nuclear localization sequence, or guide RNA extensions of instant claim 1.
These deficiencies are cured by Steinberg and Anzalone, as evidenced by Burke.
Regarding the instantly claimed R2 element enzyme comprising a reverse transcriptase domain, a nickase domain, and a targeting domain, these are considered to be endogenous domains of R2 element enzymes. As reviewed in Burke, all R2 elements recognize and nick a specific target sequence (the 28S gene sequence) via an endonuclease domain that nicks said target sequence (page 505, column 1, paragraph 2; page 508, column 1, paragraph 1). Accordingly, the nicking activity and specific targeting of R2 element enzymes is considered to read on the instantly claimed nickase domain and targeting domain. This is supported by the instant specification, as it is explicitly disclosed that the targeting domain may be a natural targeting domain (paragraph [0010]). Furthermore, as shown in Figure 1 of Burke, all R2 element enzymes also comprise a reverse transcriptase domain. Thus, the claimed R2 element enzyme domains are all considered to be endogenous R2 domains present in any R2 element enzyme.
Furthermore, as set forth above, the Gene Writer™ system of Steinberg is further disclosed to comprise a guide RNA as part of the template RNA (embodiment 448: page 69), as well as a guide RNA to target a specified genomic site (page 557, lines 3-12). The template RNA of the Gene Writer™ system is considered to read on the instantly claimed payload RNA, as the template RNA comprises a sequence that binds the Gene Writer™ polypeptide, a heterologous insert sequence (page 90, lines 5-6), and a guide RNA (embodiment 448: page 69) with a 3’ extension to include donor sequence for genome modification (Figure 8B; page 847, lines 11-13) (i.e. a pegRNA as disclosed at page 589, lines 14-15). Additionally, regarding the nuclear localization signal at the N-terminus or C-terminus of the R2 element enzyme, the Gene Writer™ system of Steinberg is further disclosed to comprise a nuclear localization signal fused to the N-terminus or C-terminus of a Gene Writer™ as described therein (i.e. encompassing a wild type R2 Gene Writer™) (Figure 29B; page 151, lines 17-24) and as instantly claimed.
Per Anzalone, prime editors are capable of introducing PAM-proximal point mutations, small insertions, and small deletions; whereas, Cas transposes/recombinases are capable of introducing large insertions (Figure 1). While these Cas transposes/recombinases disclosed in Anzalone do not specifically read on the instantly claimed Cas-R2 retrotransposon fusions, they are broadly drawn to Cas and dCas fusions to transposons for CRISPR-associated transposon-mediated genomic integration (page 836, column 1, paragraphs 2-4; page 836, column 2, paragraph 3-page 837, column 1, paragraph 1; Figure 6). The disclosure of Anzalone thus establishes that prime editing and CRISPR-associated transposon-mediated gene editing were both known in the field and were known to accomplish different editing outcomes prior to the effective filing date of the instant invention. Therefore, based on the knowledge generally available to one of ordinary skill in the art prior to the effective filing date of the instant invention, it is considered that it would have been obvious to one of ordinary skill in the art to experiment with combining these various genome editing technologies for purposes of optimizing the desired editing outcome(s), as different technologies accomplish different types of editing events (even simultaneously), as depicted in Figure 1 of Anzalone.
With regard to instant claim 16, which recites “the Cas12 protein [of the genome editing system of claim 1] is fully active or catalytically dead,” as set forth above, copending claim 2 recites that a suitable programmable DNA-binding protein for use in the system taught therein is a Cas12 protein that is a nickase, as instantly claimed.
With regard to instant claim 20, which recites “the payload RNA [of the genome editing system of claim 1] further comprises one or more of a 5’ homology region or a 3’ homology region,” as set forth above, copending claim 24 also recites that the donor construct claimed therein comprises a first and second homology region, as instantly claimed.
Finally, with regard to instant claim 37, which recites “a method of inserting a nucleic acid into a genome within a cell using a Cas9 or Cas12 fusion protein having nickase activity, wherein the method comprises supplying a Cas9 or Cas12 fusion protein to a cell, wherein the Cas9 or Cas12 fusion protein is supplied with at least one guide RNA, and a payload RNA template comprising a nucleic acid sequence, wherein the payload RNA further comprises a Cas9 or Cas12 guide RNA, wherein the RNA template is reverse transcribed by the Cas9 or Cas12 fusion protein prior to being inserted into the genome of the cell; and wherein the nucleic acid sequence is inserted into the genome of the cell; and wherein the Cas9 or Cas12 guide RNA comprises an extension with a 5’ homology sequence, a 3’ homology sequence, a 5’ untranslated region (UTR), a 3’ UTR, an insertion template, or any combination thereof,” while the copending application (i.e. claims 1, 2, and 44) also recites a method of inserting a donor polynucleotide into a target polynucleotide by supplying a composition comprising a programmable DNA-binding protein (i.e. Cas12 nickase) fused to a non-LTR retrotransposon polypeptide (i.e. R2), the copending application does not recite all the individual steps, such as reverse transcription, of the instantly claimed method.
However, this deficiency is cured by Steinberg.
As set forth above, Steinberg discloses the Gene Writer™ system comprising fusion of R2 element enzymes (which may have a nuclear localization signal fused to the N-terminus thereof) and a Cas enzyme such as a Cas9 nickase, as well as at least one guide RNA and a payload RNA comprising a protein binding element, an insertion template, and a Cas9 guide RNA, said guide RNA comprising an extension with an insertion template (i.e. a pegRNA: page 589, lines 14-15; reviewed in Anzalone), as instantly claimed. Furthermore, Steinberg discloses that the system taught therein reverse transcribes the template RNA sequence for insertion into the host genome, thereby modifying the host genome (page 90, lines 17-19), as instantly claimed. Thus, while the copending application does not recite all the individual steps of the instantly claimed method, Steinberg supplies all the additional method steps required to arrive at the instantly claimed genome editing method.
Given that both the copending and instant applications recite gene editing compositions, said compositions comprising a non-LTR retrotransposon polypeptide (such as an R2 element) with a site-specific nuclease (that may be a Cas12 nickase), and a donor polynucleotide within a retrotransposon RNA (i.e. the instantly claimed payload RNA as further disclosed by Steinberg and set forth above), and that Steinberg (as evidenced by Burke) further discloses that the guide RNA of such a gene editing system may be a part of the payload (or retrotransposon or template) RNA and may further comprise an insert template to facilitate small insertions or deletions in addition to the large insertions facilitated by R2-Cas fusion, as well as that the R2 element may comprise a nuclear localization signal fused to the N-terminus or C-terminus, 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 the copending application per the disclosure of Steinberg (as evidenced by Burke) to predictably produce a genome editing system capable of simultaneously inserting or deleting small DNA sequences and generating large insertions efficiently and precisely, as taught by Anzalone and recited at instant claim 37 and copending claim 44. One would have been motivated to make such a modification in order to receive the expected benefit of generating a genome editing system capable of inserting DNA sequences with single-nucleotide precision while simultaneously generating large insertions. Accordingly, it is considered that the copending and instant subject matter is not patentably distinct.
This is a provisional nonstatutory double patenting rejection.
Conclusion
No claims are allowed.
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/SARAH E ALLEN/Examiner, Art Unit 1637
/Jennifer Dunston/Supervisory Patent Examiner, Art Unit 1637
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