DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Election/Restrictions
Applicant’s election of claims 1-18, 54-62, and 69-80 (Group I) in the reply filed on 07/15/2026 is acknowledged. Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)).
Claims 2-10, 12, 17, 18, 54-62, and 69-80 would be withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim; however, these claims were cancelled in the amended claim set filed 07/15/2026 thereby obviating this withdrawal. Election was made without traverse in the reply filed on 07/15/2026.
Claims 82-95 were added in the amended claim set filed 07/15/2026. Applicant asserts that these claims fall within the elected group. Upon review of the application, the Examiner concurs.
Accordingly, claims 1, 11, 13-16, and 82-95 are pending and under consideration.
Priority
Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged. The earliest effective filing date to which the instant application is entitled is 03/19/2019.
Information Disclosure Statement
Receipt of information disclosure statements on 08/24/2023 and 07/15/2026 is acknowledged. The signed and initialed PTO-1449‘s have been mailed with this action.
Drawings
The drawings filed 05/24/2023 are accepted.
Specification
The lengthy specification has not been checked to the extent necessary to determine the presence of all possible minor errors. Applicant’s cooperation is requested in correcting any errors of which applicant may become aware in the specification.
Claim Objections
Claims 1, 89, 90, 92, 94, and 95 are objected to because of the following informalities:
While instant claim 1 defines several acronyms prior to their recitation (i.e. prime editing guide RNA is acronymized as PEgRNA per lines 3-4), line 8 recites the acronym “gRNA” without defining said acronym prior to reciting the same. For purposes of clarity and propriety, it would be remedial to define the acronym “gRNA” (i.e. guide RNA) prior to reciting the associated acronym. Furthermore, instant claim 1 inconsistently recites “double-stranded DNA” and “double stranded DNA” (underlined emphasis added). It would be remedial to recite either “double-stranded DNA” or “double stranded DNA” (underlined emphasis added). This issue is repeated in instant claims 90, 94, and 95, all of which also recite double stranded DNA. All of these recitations should be consistent throughout the instant claim set. Finally, instant claim 1 recites “a single strand DNA,” which does not comport with standard grammatical and/or linguistic conventions. For purposes of comporting with such conventions, it would be remedial to amend the instant claim language to recite “a single stranded DNA” or “a single strand of DNA” (bolded emphasis added), or another appropriate phrase. These are merely examples set forth by the Examiner and are not intended to be limiting.
With further regard to instant claim 1, the repeated recitation of “single strand DNA” (bolded emphasis added) is grammatically improper. It would be remedial to amend the instant claim language to be grammatically proper, for example by reciting “single stranded DNA.” This is merely an example set forth by the Examiner and is not intended to be limiting.
Instant claim 89 recites an acronym “MS2cp” without defining said acronym prior to reciting the same. For purposes of clarity and propriety, it would be remedial to define the acronym “MS2cp” (i.e. MS2 coat protein) prior to reciting the associated acronym.
Claim 92 recites the acronym “mRNA” without first defining said acronym. For purposes of clarity and propriety, it would be remedial to define the acronym “mRNA” (i.e. messenger RNA) prior to reciting the associated acronym.
With regard to claim 95, which recites “a ngRNA spacer sequence” (bolded emphasis added), the article “a” is grammatically improper when preceding words articulated with an initial vowel sound. While “ngRNA” is spelled such that it recites a consonant letter “n,” the pronunciation of the “n” in “ngRNA” nonetheless begins with a vowel sound. It would be remedial to update the instant claim language to be grammatically proper.
Appropriate correction is required.
Claim Rejections - 35 USC § 112(a) – Written Description
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 13 and 15 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Claims 13 and 15 are separately drawn to a set of amino acid sequences corresponding to a napDNAbp (claim 13) and a reverse transcriptase (claim 15), wherein the napDNAbp is defined as comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 137 and wherein the reverse transcriptase is defined as comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 139. The rejected claims thus comprise a set of variable sequences that encompass a large number of variable residues, wherein each variation must correspond to a functional napDNAbp or reverse transcriptase, respectively.
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 numerous sequences corresponding to both napDNAbps (see paragraphs [0026], [0311], [0527], [0528], and [0529] for examples) and reverse transcriptases (see paragraphs [0027], [0527], [0528], and [0529] for examples) suitable for use in the prime editing compositions claimed therein. However, no description is provided of sequences having at least 80% sequence identity with the disclosed sequences, wherein such variable sequences correspond to a functional napDNAbp or reverse transcriptase, respectively. While the instant specification discloses that conservative amino acid substitutions are likely to have minor effects on protein structure and are likely to be well tolerated without compromising function (paragraph [0359]), the instant claim language does not limit this variation to conservative substitutions only.
Even if one accepts that the examples described in the specification meet the claim limitations of the rejected claims with regard to structure and function, the examples are only representative of the disclosed sequences and of conservative substitutions thereof. The results are not necessarily predictive of sequences having at least 80% sequence identity with the disclosed sequences, including sequences with non-conservative substitutions. Thus, it is impossible for one to extrapolate from the examples described herein those sequences 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 in that it does not describe a set of variable amino acid sequences, wherein all variations having at least 80% identity to the instantly claimed sequences retain the functions of a napDNAbp and a reverse transcriptase, respectively. Furthermore, as set forth above, although the instant specification discloses that conservative amino acid substitutions are likely to have minor effects on protein structure and are likely to be well tolerated without compromising function (paragraph [0359]), the instant claim language does not limit this variation to conservative substitutions only. As is known to those of ordinary skill in the art, while conservative substitutions are more likely to be accepted, other substitutions may be non-conservative, which are less likely to be accepted and are more likely to alter structure/function (reviewed in Goldstein and Pollock, 2016; see entire document).
Therefore, the skilled artisan would have reasonably concluded applicants were not in possession of the claimed invention for claims 13 and 15.
Claim Rejections - 35 USC § 112(a) - Enablement
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, 11, 13-16, and 82-95 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for editing a double-stranded DNA sequence by contacting the double-stranded DNA sequence with a complex comprising a prime editor comprising a napDNAbp that is a Cas nickase and an RNA or DNA-dependent DNA polymerase, directed by a prime editing guide RNA (PEgRNA), does not reasonably provide enablement for editing a double-stranded DNA sequence by contacting the double-stranded DNA sequence with a complex comprising a prime editor comprising any napDNAbp and any polymerase capable of nicking a non-target strand of the double-stranded DNA sequence, directed by a PEgRNA. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention commensurate in scope with these claims.
Enablement is considered in view of the Wands factors (MPEP 2164.01(A)). These include: the breadth of the claims, the nature of the invention, the state of the prior art, the level of one of ordinary skill, the level of predictability in the art, the amount of direction provided by the inventor, the existence of working examples, and the quantity of experimentation needed to make or use the invention. All of the Wands factors have been considered with regard to the instant claims, with the most relevant factors discussed below.
Nature of the invention: The invention is broadly drawn to a method of editing a double-stranded DNA sequence, said method comprising contacting the double-stranded DNA sequence with a complex comprising a prime editor (comprising a napDNAbp capable of nicking a non-target strand of the double-stranded DNA sequence to produce a single stranded DNA having a 3’ end and a polymerase) and a PEgRNA (comprising a spacer sequence capable of targeting the napDNAbp to a target strand of the double stranded DNA sequence, a gRNA core capable of binding to the napDNAbp, and a DNA extension arm). The invention therefore embraces any napDNAbp capable of nicking a non-target strand of the double-stranded DNA sequence to produce a single stranded DNA having a 3’ end when directed by a PEgRNA, both of which must function in the instantly claimed method with the instantly claimed machinery. Furthermore, the invention embraces any polymerase, which includes RNA and DNA-dependent DNA polymerases, as well as RNA polymerases that synthesize RNA.
Breadth of the claims: The claims broadly encompass a method of editing a double-stranded DNA sequence, wherein said method utilizes any napDNAbp capable of nicking a non-target strand of the double-stranded DNA sequence to produce a single stranded DNA having a 3’ end when directed by a PEgRNA.
The complex nature of the subject matter of this invention is greatly exacerbated by the breadth of the claims.
Guidance of the specification and existence of working examples: While the instant specification envisions replacing the SpCas9 nickase of the instant invention with another napDNAbp such as zinc finger nucleases (ZFN) or transcription activator-like effector nucleases (TALEN) (paragraph [0079]), none of these ideas are reduced to practice. The instant specification sets forth that the person having ordinary skill in the art will appreciate that any suitable napDNAbp may be used in the invention claimed herein. However, the examples of the instant specification are drawn solely to editing methods comprising Cas napDNAbps. While Example 16 expands the targeting scope of prime editing by utilizing Staphylococcus aureus CRISPR-Cas as the RNA-guided DNA binding protein, this Example does not set forth how a ZFN or a TALEN would function in a system that requires a specialized guide RNA per the instant claim set. No description is provided of actually reducing these ideas to practice.
Furthermore, while the instant claim language is drawn to any polymerase species, the instant specification is drawn to DNA polymerases (see paragraph [0010]), which synthesize DNA for insertion. While the instant specification contemplates the use of “alternative polymerases” (paragraph [1474]), there are no working examples disclosed of any polymerase other than a reverse transcriptase, which synthesizes DNA from an RNA template (reviewed in Coffin and Fan, 2016: see entire document). Given that the instantly claimed methods are drawn to a method for editing a double-stranded DNA sequence, it is unclear how an RNA polymerase that synthesizes RNA would be suitable for use in the instantly claimed methods.
Predictability and state of the art: As is known to those of ordinary skill in the art, neither ZFNs nor TALENs utilize guide RNAs to target their specified sequences (reviewed in Gaj et al., 2013: see entire document). Although both ZFNs and TALENs may be engineered to function as nickases (i.e. to nick a non-target strand), these nickase species also do not utilize guide RNAs to targeted their specified sequences (reviewed in Gaj et al., 2013: see entire document; TALE nickases taught in Luo et al., 2016: see entire document).
Furthermore, as is known to those of ordinary skill in the art, DNA polymerases include reverse transcriptases, which synthesize DNA from an RNA template (reviewed in Coffin and Fan, 2016: see entire document). Certain reverse transcriptases, such as the Moloney murine leukemia virus reverse transcriptase (MMLV-RT) is also capable of using DNA as a template to synthesize DNA (reviewed in Ohtsubo et al., 2017: see entire document). The instantly claimed methods are drawn to a method for editing a double-stranded DNA sequence; accordingly, it is considered that the polymerase claimed herein must generate DNA for insertion into the double-stranded DNA sequence. It is unclear how an RNA polymerase that synthesizes RNA would be suitable for use in the instantly claimed methods.
Amount of experimentation necessary: The quantity of experimentation required to carry out the full scope of the claimed method is large. One could not solely rely upon guidance provided in the instant disclosure or in the prior art. As set forth above, while the instant specification envisions alternative napDNAbps other than Cas nickases, neither the instant specification nor the prior art disclose methods of using these alternative napDNAbps in the instant invention, which requires a guide RNA per the instant claim language. In fact, the guide RNA is crucial to the technology claimed herein, as the guide RNA must escort the napDNAbp to the targeted site as well as provide the intended edit for said site.
Furthermore, given that the instantly claimed methods are drawn to a method for editing a double-stranded DNA sequence, it is considered that the polymerase claimed herein must generate DNA for insertion into the double-stranded DNA sequence. It is unclear how an RNA polymerase that synthesizes RNA would be suitable for use in the instantly claimed methods.
In view of the breadth of the claims and the lack of guidance provided by the specification as well as the unpredictability of the art, the skilled artisan would have required an undue amount of experimentation to make and/or use the claimed invention. Therefore, claims 1, 11, 13-16, and 82-95 are not considered to be fully enabled by the instant disclosure.
The Examiner notes that instant claims 11, 13, 14, and 15 are included in the rejection. While instant claims 11 and 13 limit the napDNAbp to a Cas9 nickase with a specified sequence, the instant claims do not limit the instantly claimed polymerase. Additionally, while instant claims 14 and 15 limit the polymerase to a reverse transcriptase with a specified sequence, the instant claims do not limit the instantly claimed napDNAbp.
Claims 90-93 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for editing double stranded DNA in a cell in vitro or in a cell in an animal model in vivo, does not reasonably provide enablement for editing double stranded DNA in a cell in a human in vivo. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention commensurate in scope with these claims.
Enablement is considered in view of the Wands factors (MPEP 2164.01(A)). These include: the breadth of the claims, the nature of the invention, the state of the prior art, the level of one of ordinary skill, the level of predictability in the art, the amount of direction provided by the inventor, the existence of working examples, and the quantity of experimentation needed to make or use the invention. All of the Wands factors have been considered with regard to the instant claims, with the most relevant factors discussed below.
Nature of the invention: Claims 90-93 are broadly drawn to a method of editing double-stranded DNA in a cell, wherein the cell may be in a human (as recited at instant claim 91). The claims thus broadly read on in vivo genome editing in a human being.
Breadth of the claims: As set forth above, claims 90-93 are broadly drawn to a method of editing double-stranded DNA in a cell, wherein the cell may be in a human (as recited at instant claim 91). While instant claims 92 and 93 further limit the manner of delivery, they nonetheless still read on in vivo genome editing in a human being, which is an unpredictable and underdeveloped topic in the art. The instant claims embrace any napDNAbp (as set forth above), including Cas nickases such as Cas9 nickase for use in said method of editing double-stranded DNA in a cell.
The complex nature of the subject matter of this invention is greatly exacerbated by the breadth of the claims.
Guidance of the specification and existence of working examples: While the instant specification discloses in vitro editing of human cells (see Figures 17A-17C; paragraphs [1152], [1164], [1165], and [1168]-[1191]), which represent promising advances in gnome editing capabilities, none of these examples practice in vivo genome editing of a cell in a human, as is instantly claimed. Particularly, none of these examples practice in vivo genome editing of a cell in a human using a Cas nickase such as a Cas9 nickase.
Predictability and state of the art: As set forth above, while the instant specification discloses in vitro prime editing of human cells, it is silent as to in vivo prime editing of cells such as human cells. The examples set forth in the instant specification (as set forth above) are all drawn to editing via a Cas nickase such as a Cas9 nickase. However, applying CRISPR machinery to human beings is an underdeveloped and unpredictable topic in the art. As reviewed in Behr et al., 2021 and Liu et al., 2021 (which post-date the effective filing date but are applied for evidentiary purposes only), there are numerous hurdles to overcome before in vivo editing with CRISPR machinery such as a Cas9 nickase is commonplace. These hurdles include, but are not limited to, editing efficiency (discussed in section “Editing efficiency” of Liu et al., 2021), effective delivery of the editing machinery (discussed in section 6 of Behr et al., 2021 and section “Delivering methods” of Liu et al., 2021), off-target effects (discussed in section “Off-target effects” of Liu et al., 2021), and immunogenicity (discussed in section “Immunogenicity” of Liu et al., 2021).
Amount of experimentation necessary: The quantity of experimentation required to carry out the full scope of the claimed methods is large. One could not solely rely upon guidance provided in the instant disclosure or in the prior art. As set forth above, the prior art teaches that in vivo CRISPR-mediated editing is an underdeveloped and unpredictable topic in the art. Thus, while these systems may be functionally validated in vitro, as set forth in the instant specification, these results do not offer any guarantee of success of the same methods when applied in vivo.
In view of the breadth of the claims and the lack of guidance provided by the specification as well as the unpredictability of the art, the skilled artisan would have required an undue amount of experimentation to make and/or use the claimed invention. Therefore, claims 90-93 are not considered to be fully enabled by the instant disclosure.
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, 11, 14, 16, 82-90, 92, and 93 are rejected under 35 U.S.C. 103 as being unpatentable over WO 2018/049168 A1 (hereinafter Fraser; as cited in the IDS filed 08/24/2023) in view of Komor et al., 2017 (hereinafter Komor; as cited in the IDS filed 08/24/2023).
With regard to claim 1, which recites “a method for editing a double-stranded DNA sequence, the method comprising: contacting the double-stranded DNA sequence with a complex comprising a prime editor and a prime editing guide RNA (PEgRNA), wherein the prime editor comprises (i) a nucleic acid programmable DNA binding protein (napDNAbp) capable of nicking a non-target strand of the double stranded DNA sequence to produce a single strand[ed] DNA having a 3’ end and (ii) a polymerase, and wherein the PEgRNA comprises: (i) a spacer sequence capable of targeting the napDNAbp to a target strand of the double stranded DNA sequence, (ii) a gRNA core capable of binding to the napDNAbp, and (iii) a DNA extension arm comprising a DNA synthesis template encoding the desired nucleotide change and a primer binding site capable of hybridizing to the single strand[ed] DNA having the 3’ end,” Fraser discloses methods for high-efficiency genome editing, wherein the method comprises providing retron-guide RNA cassettes (abstract). The retron-guide RNA cassettes of Fraser read on the instantly claimed PEgRNA, as they comprise a retron transcript (i.e. the extension arm of the claim) and a guide RNA molecule comprising a spacer complementary to a first strand and a guide RNA core capable of binding to the Cas napDNAbp taught therein (paragraphs [0003], [0006], [0009], [0010], and [0127]). Fraser explicitly discloses that the retron transcript of the retron-guide RNA cassettes taught therein is capable of self-priming reverse transcription by a reverse transcriptase (paragraph [0020]). The reverse transcriptase of Fraser (see paragraphs [0106], [0147], [0148], and [0154]) reads on the instantly claimed polymerase (see paragraph [0010] of the instant specification), while the Cas nickase disclosed therein (see paragraphs [0066], [0138], [0142], and [0143]) read on the instantly claimed napDNAbp capable of nicking a strand of the double stranded DNA sequence to produce a single stranded DNA having a 3’ end. Thus, Fraser discloses each and every limitation of the instantly claimed method with the exception of specifically nicking the non-target strand. This deficiency is cured by Komor.
While the disclosure of Fraser is drawn to coordinated actions of a Cas nickase (paragraphs [0066], [0138], [0142], and [0143]) and a reverse transcriptase (paragraphs [0020], [0106], [0147], [0148], and [0154]), Cas nickase activity has been coordinated with other effector proteins. Komor discloses base editors, which comprise a nickase Cas9 such as nSpCas9 and nSaCas9 fused to a base editing enzyme such as APOBEC1, wherein the Cas9 nickase nicks the non-edited strand in order to facilitate base editing of the targeted residue in the edited (i.e. targeted) strand (Figure 2). While the disclosure of Fraser and Komor differ in their effects, both disclosures are drawn to coordinated actions of a Cas nickase and an effector protein for purposes of editing a targeted nucleic acid. Therefore, based on these disclosures, one of ordinary skill in the art would have been motivated, prior to the effective filing date of the instant application, to apply the principles disclosed in Komor to the methods of Fraser such that the Cas nickase disclosed therein specifically nicks the non-targeted strand in order to specifically edit the targeted strand, as set forth in greater detail below.
With regard to claim 11, which recites “the napDNAbp [of the method of claim 1] is a Cas9 nickase (nCas9),” as set forth above, Fraser discloses coordinated actions of a Cas nickase and a reverse transcriptase, wherein the Cas nickase is specifically a Cas9 nickase (paragraph [0143]).
With regard to claim 14, which recites “the polymerase [of the method of claim 1] is a reverse transcriptase,” as set forth above, Fraser discloses coordinated actions of a Cas nickase and a reverse transcriptase (paragraphs [0009], [0106], [0147], [0148], and [0154]).
With regard to claim 16, which recites “the DNA extension arm [of the method of claim 1] is at the 3’ end of the PEgRNA,” Fraser further discloses that the retron-guide RNA cassette taught therein may be structured such that the gRNA coding region is located 3’ or 5’ of the retron (paragraphs [0010] and [0102]).
With regard to claim 82, which recites “the prime editor [of the method of claim 1] is a fusion protein,” as set forth above, Fraser discloses coordinated actions of a Cas nickase and a reverse transcriptase for purposes of editing a nucleic acid sequence. However, Fraser does not explicitly disclose fusion of the Cas nickase and the reverse transcriptase taught therein. This deficiency is cured by Komor. Komor discloses base editors, which comprise a nickase Cas9 such as nSpCas9 and nSaCas9 fused to a base editing enzyme such as APOBEC1 (Figure 2). While the disclosure of Fraser and Komor differ in their effects, both disclosures are drawn to coordinated actions of a Cas nickase and an effector protein for purposes of editing a targeted nucleic acid. Therefore, based on these disclosures, one of ordinary skill in the art would have been motivated, prior to the effective filing date of the instant application, to apply the principles disclosed in Komor to the methods of Fraser such that the Cas nickase disclosed therein is fused to the reverse transcriptase disclosed therein.
With regard to claim 83, which recites “the napDNAbp [of the method of claim 82] is N-terminal of the polymerase in the fusion protein,” as set forth above, Fraser and Komor collectively disclose a fusion protein comprising a Cas nickase and a reverse transcriptase. However, neither specifically teaches the architecture of said fusion protein. Given that the fusion protein collectively disclosed by Fraser and Komor comprises only two protein components, there are only two possible arrangements: one in which the Cas nickase is N-terminal of the reverse transcriptase in the fusion protein, and one in which the Cas nickase is C-terminal of the reverse transcriptase in the fusion protein. Per MPEP § 2144.04(VI)(C), it is not inventive to rearrange parts. Given that there are only a small, finite number of possible arrangements, it is not considered inventive to merely rearrange these components.
With regard to claim 85, which recites “the polymerase [of the method of claim 1] is not connected to the napDNAbp in a fusion protein,” as set forth above, Fraser discloses coordinated actions of a Cas9 nickase and a reverse transcriptase for purposes of editing a nucleic acid sequence. Fraser further discloses that the reverse transcriptase and Cas effector disclosed therein may be provided separately (paragraphs [0026] and [0027]).
With regard to claim 90, which recites “the double stranded DNA [of the method of claim 1] is in a cell,” Fraser discloses that the methods taught therein may be practiced within a genome of a host cell, wherein the host cell is transformed with a vector carrying the machinery set forth above and is subsequently edited (paragraph [0024]).
With regard to claim 94, which recites “the method of claim 1, further comprising contacting the double stranded DNA with a complex comprising the prime editor and a nicking guide RNA (ngRNA),” Fraser further discloses that providing at least two DNA-targeting RNAs targeting opposite DNA strands facilitates a double-nicked double-strand break that favors HDR and decreases the frequency of insertion/deletion mutations at off-target DNA sites (paragraph [0143]). Per Fraser, two nickases of the invention disclosed therein may be utilized to create two single-stranded breaks on opposite strands of a target DNA, as set forth above (paragraph [0066]).
With regard to claim 95, which recites “the ngRNA [of the method of claim 94] comprises a[n] ngRNA spacer sequence capable of targeting the napDNAbp to the non-target strand of the double-stranded DNA sequence,” as set forth above, Fraser further discloses that providing at least two DNA-targeting RNAs targeting opposite DNA strands facilitates a double-nicked double-strand break that favors HDR and decreases the frequency of insertion/deletion mutations at off-target DNA sites (paragraph [0143]). Per Fraser, two nickases of the invention disclosed therein may be utilized to create two single-stranded breaks on opposite strands of a target DNA (paragraph [0066]). The DNA-targeting RNAs of Fraser read on the guide RNAs of Fraser, which are disclosed to comprise a crRNA spacer sequence that is complementary to a target nucleic acid sequence (paragraph [0127]).
Given that Fraser discloses coordinated actions of a Cas nickase and a reverse transcriptase (which may be provided separately), wherein the reverse transcriptase reverse transcribes a retron-guide RNA cassette that is subsequently inserted into the genome of a host cell, and that Komor discloses coordinated actions of a Cas9 nickase and a base editing enzyme, wherein the nickase nicks the non-edited strand such that the fused base editing enzyme may edit the target strand, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to apply the principles disclosed in Komor to the methods of Fraser such that the Cas nickase disclosed therein specifically nicks the non-targeted strand to predictably specifically edit the targeted strand. One would have been motivated to make such a modification in order to receive the expected benefit of specifically editing the targeted strand. Furthermore, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to utilize dual gRNAs that predictably facilitate nicking of both the target and non-target strand. One would have been motivated to make such a modification in order to receive the expected benefit of favoring HDR and decreasing the frequency of insertion/deletion mutations at off-target DNA sites.
Claim 13 is rejected under 35 U.S.C. 103 as being unpatentable over WO 2018/049168 A1 (hereinafter Fraser; as cited in the IDS filed 08/24/2023) in view of Komor et al., 2017 (hereinafter Komor; as cited in the IDS filed 08/24/2023) as applied to claim 1 above, and further in view of US 2016/0289673 A1 (hereinafter Huang).
The combined disclosures of Fraser and Komor are described above and applied as before. However, these disclosures do not teach the napDNAbp sequence of instant claim 13.
With regard to claim 13, which recites “the napDNAbp [of the method of claim 1] comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 137,” as set forth above, Fraser discloses that a Cas9 nickase may be used in the editing methods disclosed therein. However, Fraser is silent as to the sequence of said Cas9 nickase. This deficiency is cured by Huang.
Huang discloses that an exemplary nicking Cas9 protein is Cas9 H840A, which corresponds to SEQ ID NO: 6 taught therein (paragraphs [0077] and [0080]). As shown in the alignment of the Appendix, SEQ ID NO: 6 of Huang comprises 100% identity to instant SEQ ID NO: 137.
Given that Fraser and Komor collectively disclose the method of claim 1, wherein a Cas9 nickase coordinates activity with a reverse transcriptase to insert a desired edit into a host cell’s genome, and that Huang discloses that Cas9 H840A is an exemplary Cas9 nickase with the sequence of SEQ ID NO: 6 taught therein, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to utilize the Cas9 H840A disclosed in Huang in the methods collectively disclosed by Fraser and Komor to predictably nick the host cell genome and insert a desired edit into said host cell genome. One would have been motivated to make such a modification in order to receive the expected benefit of nicking the host cell genome and inserting a desired edit into said host cell genome.
Claim 15 is rejected under 35 U.S.C. 103 as being unpatentable over WO 2018/049168 A1 (hereinafter Fraser; as cited in the IDS filed 08/24/2023) in view of Komor et al., 2017 (hereinafter Komor; as cited in the IDS filed 08/24/2023) as applied to claims 1 and 14 above, and further in view of Ohtsubo et al., 2017 (hereinafter Ohtsubo), Yin et al., 2018 (hereinafter Yin), and UniProt Entry P03355 (hereinafter UniProt; first entered 07/21/1986).
The combined disclosures of Fraser and Komor are described above and applied as before. However, these disclosures do not teach the reverse transcriptase sequence of instant claim 15.
With regard to claim 15, which recites “the reverse transcriptase [of the method of claim 14] comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 139,” as set forth above, Fraser discloses that a reverse transcriptase may be used in the editing methods disclosed therein. However, Fraser is silent as to the sequence of said reverse transcriptase. This deficiency is cured by Ohtsubo, Yin, and Uniprot, as set forth below.
Ohtsubo teaches that the Moloney murine leukemia virus (MMLV) reverse transcriptase (MMLV-RT) is widely used for cDNA synthesis (i.e. RNA to DNA) but also has DNA polymerase activity when using DNA as a template (page 1, paragraph 3 of the introduction). Per Yin, adding DNA residues to CRISPR guide RNAs significantly reduces off-target genome editing and reduces the associated cost (abstract). Therefore, prior to the effective filing date of the instant invention, one of ordinary skill in the art would have been motivated to generate a guide RNA per the teachings of Yin that comprises DNA residues. Therefore, the reverse transcriptase disclosed in Fraser must be capable of synthesizing DNA from both DNA and RNA in order to utilize both chimeric guide RNAs and standard guide RNAs efficiently. As set forth above, Ohtsubo teaches that MMLV-RT is uniquely suited to both RNA to DNA synthesis and DNA to DNA synthesis. Accordingly, one of ordinary skill in the art would have been motivated to utilize MMLV-RT in the methods of Fraser. UniProt teaches the sequence of MMLV-RT, which comprises 98.8% sequence identity to instant SEQ ID NO: 139, as shown in the alignment of the Appendix.
Given that Fraser and Komor collectively disclose the method of claim 14, wherein a Cas9 nickase coordinates activity with a reverse transcriptase to insert a desired edit into a host cell’s genome, that Ohtsubo discloses that MMLV-RT (as taught by UniProt) is uniquely suited to both RNA to DNA synthesis and DNA to DNA synthesis, and that Yin teaches that chimeric guide RNAs comprising both DNA and RNA offer scientific and practical advantages (i.e. reduced off-target effects and reduced cost to practice), it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to utilize MMLV-RT as the reverse transcriptase in the methods collectively disclosed by Fraser and Komor to predictably produce DNA for targeted genome insertion using both standard and chimeric guide RNAs. One would have been motivated to make such a modification in order to receive the expected benefit of producing DNA for targeted genome insertion using both standard and chimeric guide RNAs.
Claims 84, 86-89, 92, and 93 are rejected under 35 U.S.C. 103 as being unpatentable over WO 2018/049168 A1 (hereinafter Fraser; as cited in the IDS filed 08/24/2023) in view of Komor et al., 2017 (hereinafter Komor; as cited in the IDS filed 08/24/2023) as applied to claims 1, 83, 85, and 90 above, and further in view of WO 2019/005884 (hereinafter Zhang; as cited in the IDS filed 08/24/2023).
The combined disclosures of Fraser and Komor are described above and applied as before. However, these disclosures do not teach the linker of instant claim 84, the RNA-binding protein and recruitment domain thereof of instant claims 86-89, or the delivery methods of instant claims 92-93.
With regard to claim 84, which recites “the prime editor [of the method of claim 83] further comprises a peptide linker between the napDNAbp and the polymerase,” as set forth above, Fraser and Komor collectively disclose the method of claim 83. However, neither Fraser nor Komor disclose the peptide linker of instant claim 84. This deficiency is cured by Zhang.
Zhang discloses methods and compositions for targeting and editing nucleic acids, particularly RNA targeted and edited via a Cas13 fusion protein comprising a Cas13 fused to an editing enzyme such as ADAR (abstract; paragraph [0047]; Figure 2). While the methods of Zhang are drawn to RNA editing and not DNA editing, Zhang nonetheless discloses coordinated activity of a Cas enzyme and an editing enzyme, as in the instant application, Fraser, and Komor. Zhang further discloses that the fusion proteins taught therein may be linked via a peptide linker (paragraphs [0200] and [0201]). Thus, Zhang establishes that Cas proteins may be fused to effector proteins via a peptide linker, as instantly claimed.
With regard to claim 86, which recites “the PEgRNA further comprises an RNA-binding protein recruitment domain,” as set forth above, the retron-guide cassette of Fraser reads on the instantly claimed PEgRNA. Zhang further discloses that the guide molecule taught therein comprises an aptamer sequence capable of binding to an RNA-binding adaptor protein such as MS2 (paragraphs [0011], [0315], and [0316), which reads on the instantly claimed RNA-binding protein recruitment domain.
With regard to claim 87, which recites “the RNA-binding protein recruitment domain [of the method of claim 86] is an MS2 aptamer,” as set forth above, Zhang further discloses that the guide molecule taught therein comprises an aptamer sequence capable of binding to an RNA-binding adaptor protein such as MS2 (paragraphs [0011], [0315], and [0316).
With regard to claim 88, which recites “the polymerase [of the method of claim 85] is fused to an RNA-binding protein domain,” Zhang further discloses that the adenosine deaminase effector taught therein may be fused to RNA-binding protein MS2 coat protein (paragraphs [0315] and [0316]). While the adenosine deaminase effector of Zhang does not read on the instantly claimed polymerase, the disclosure of Zhang is applied to establish that principles of coordinating activity of a Cas protein and an editing enzyme, as in the instant application, Fraser, and Komor. Therefore, Zhang teaches that the effector/editing enzyme taught therein may be fused to an RNA-binding protein such as MS2 coat protein for purposes of implementing orthogonal gene editing (paragraphs [0315], [0316], and [0325]).
With regard to claim 89, which recites “the polymerase [of the method of claim 86] is fused to an MS2cp domain,” as set forth above, Zhang further discloses that the adenosine deaminase effector taught therein may be fused to RNA-binding protein MS2 coat protein for purposes of implementing orthogonal gene editing (paragraphs [0315], [0316], and [0325]).
With regard to claim 92, which recites “the method of claim 90, further comprising delivering an mRNA encoding the prime editor into the cell,” as set forth above, Fraser and Komor collectively disclose the instantly claimed prime editor. While Fraser discloses that the reverse transcriptase and Cas effector taught therein may be expressed from a vector or plasmid (paragraphs [0026], [0027], and [0145]), Fraser does not explicitly disclose that these components are delivered as an mRNA. This deficiency is cured by Zhang, which discloses that the editing compositions disclosed therein (including Cas-enzyme fusions) may be delivered as mRNA molecules encoding all of the components thereof (paragraphs [0318] and [0319]).
With regard to claim 93, which recites “the mRNA and the pegRNA [of the method of claim 92] are delivered via a lipid nanoparticle,” Zhang further discloses that the mRNA encoding the components of the system taught therein (and set forth above) may be delivered via one or more lipid nanoparticles (paragraphs [0318] and [0319]).
Given that Fraser and Komor collectively disclose the method of instant claim 1, wherein a Cas protein coordinates its activity with an enzyme that effects a nucleotide edit, and that Zhang also discloses a method of effecting a nucleotide edit, wherein a Cas protein coordinates its activity with an enzyme, specifically wherein the guide RNA associated with said Cas protein is fused to an MS2 aptamer that facilitates orthogonal gene editing by also fusing the MS2 coat protein to the enzyme that effects said nucleotide edit and specifically wherein the components may be delivered as RNA via lipid nanoparticles, 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 Fraser and Komor to comprise an MS2 aptamer bound to the guide RNA taught therein to predictably facilitate orthogonal gene editing by also fusing the MS2 coat protein to the enzyme that effects said nucleotide edit. One would have been motivated to make such a modification in order to receive the expected benefit of facilitating orthogonal gene editing by also fusing the MS2 coat protein to the enzyme that effects said nucleotide edit.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 1, 11, 13-16, 82-90, and 92-95 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 8-13, and 25 of U.S. Patent No. 11,447,770 B2 (as cited in the IDS filed 08/24/2023) in view of WO 2018/049168 A1 (hereinafter Fraser; as cited in the IDS filed 08/24/2023), Komor et al., 2017 (hereinafter Komor; as cited in the IDS filed 08/24/2023), US 2016/0289673 A1 (hereinafter Huang), Ohtsubo et al., 2017 (hereinafter Ohtsubo), Yin et al., 2018 (hereinafter Yin), UniProt Entry P03355 (hereinafter UniProt; first entered 07/21/1986), and WO 2019/005884 (hereinafter Zhang; as cited in the IDS filed 08/24/2023).
Claim 1 of patent ‘770 is drawn to a method for site-specific modification of a double-stranded target DNA sequence, said method comprising a fusion protein comprising a nucleic acid programmable DNA binding protein such as a Cas9 nickase and a reverse transcriptase, as well as a prime editing guide RNA comprising a spacer, a gRNA core, an extension arm, and a primer binding site, wherein contacting the double-stranded DNA target sequence with said composition nicks the second strand, thereby freeing a 3’ end at the nick site and facilitating insertion of a sequence synthesized from the DNA synthesis template of the prime editing guide RNA.
Claims 8-10 further limit the Cas9 nickase claimed therein to an H840A nickase mutant, while claim 25 further limits the Cas9 nickase to one derived from Streptococcus pyogenes. Claims 11 and 12 further limit the reverse transcriptase claimed therein to an MMLV reverse transcriptase. Claim 13 further limits the method to being practiced in a cell.
In comparison, instant claim 1 is also drawn to a method for site-specific modification of a double-stranded target DNA sequence utilizing the same machinery. Instant claim 11 further limits the napDNAbp of the instant invention to a Cas9 nickase, as in patent ‘770. Instant claim 14 further limits the polymerase of the instant invention to a reverse transcriptase, as in patent ‘770. Instant claim 82 further limits the prime editor to a fusion protein, as in patent ‘770. Instant claim 90 further limits the method to being practiced in a cell.
While the copending application is silent as to the instantly claimed sequences, as set forth above, the sequence of the napDNAbp of instant claim 13 is drawn to an H840A Cas9 mutant, which is a known nickase and comprises the entirety of SEQ ID NO: 137, as disclosed in Huang. Furthermore, as set forth above, the sequence claimed at instant claim 15 is drawn to an MMLV-RT sequence per UniProt. See the alignments of the Appendix.
With regard to claim 16, which recites “the DNA extension arm [of the method of claim 1] is at the 3’ end of the PEgRNA,” as set forth above, Fraser and Komor collectively disclose the method of instant claim 1. Fraser further discloses that the retron-guide RNA cassette taught therein may be structured such that the gRNA coding region is located 3’ or 5’ of the retron (paragraphs [0010] and [0102]).
With regard to claim 83, which recites “the napDNAbp [of the method of claim 82] is N-terminal of the polymerase in the fusion protein,” as set forth above, Fraser and Komor collectively disclose a fusion protein comprising a Cas nickase and a reverse transcriptase. However, neither specifically teaches the architecture of said fusion protein. Given that the fusion protein collectively disclosed by Fraser and Komor comprises only two protein components, there are only two possible arrangements: one in which the Cas nickase is N-terminal of the reverse transcriptase in the fusion protein, and one in which the Cas nickase is C-terminal of the reverse transcriptase in the fusion protein. Per MPEP § 2144.04(VI)(C), it is not inventive to rearrange parts. Given that there are only a small, finite number of possible arrangements, it is not considered inventive to merely rearrange these components.
With regard to claim 84, which recites “the prime editor [of the method of claim 83] further comprises a peptide linker between the napDNAbp and the polymerase,” as set forth above, Fraser and Komor collectively disclose the method of claim 83. However, neither Fraser nor Komor disclose the peptide linker of instant claim 84. This deficiency is cured by Zhang.
Zhang discloses methods and compositions for targeting and editing nucleic acids, particularly RNA targeted and edited via a Cas13 fusion protein comprising a Cas13 fused to an editing enzyme such as ADAR (abstract; paragraph [0047]; Figure 2). While the methods of Zhang are drawn to RNA editing and not DNA editing, Zhang nonetheless discloses coordinated activity of a Cas enzyme and an editing enzyme, as in the instant application, Fraser, and Komor. Zhang further discloses that the fusion proteins taught therein may be linked via a peptide linker (paragraphs [0200] and [0201]). Thus, Zhang establishes that Cas proteins may be fused to effector proteins via a peptide linker, as instantly claimed.
With regard to claim 85, which recites “the polymerase [of the method of claim 1] is not connected to the napDNAbp in a fusion protein,” as set forth above, Fraser discloses coordinated actions of a Cas9 nickase and a reverse transcriptase for purposes of editing a nucleic acid sequence. Fraser further discloses that the reverse transcriptase and Cas effector disclosed therein may be provided separately (paragraphs [0026] and [0027]).
With regard to claim 86, which recites “the PEgRNA further comprises an RNA-binding protein recruitment domain,” as set forth above, the retron-guide cassette of Fraser reads on the instantly claimed PEgRNA. Zhang further discloses that the guide molecule taught therein comprises an aptamer sequence capable of binding to an RNA-binding adaptor protein such as MS2 (paragraphs [0011], [0315], and [0316), which reads on the instantly claimed RNA-binding protein recruitment domain.
With regard to claim 87, which recites “the RNA-binding protein recruitment domain [of the method of claim 86] is an MS2 aptamer,” as set forth above, Zhang further discloses that the guide molecule taught therein comprises an aptamer sequence capable of binding to an RNA-binding adaptor protein such as MS2 (paragraphs [0011], [0315], and [0316).
With regard to claim 88, which recites “the polymerase [of the method of claim 85] is fused to an RNA-binding protein domain,” Zhang further discloses that the adenosine deaminase effector taught therein may be fused to RNA-binding protein MS2 coat protein (paragraphs [0315] and [0316]). While the adenosine deaminase effector of Zhang does not read on the instantly claimed polymerase, the disclosure of Zhang is applied to establish that principles of coordinating activity of a Cas protein and an editing enzyme, as in the instant application, Fraser, and Komor. Therefore, Zhang teaches that the effector/editing enzyme taught therein may be fused to an RNA-binding protein such as MS2 coat protein for purposes of implementing orthogonal gene editing (paragraphs [0315], [0316], and [0325]).
With regard to claim 89, which recites “the polymerase [of the method of claim 86] is fused to an MS2cp domain,” as set forth above, Zhang further discloses that the adenosine deaminase effector taught therein may be fused to RNA-binding protein MS2 coat protein for purposes of implementing orthogonal gene editing (paragraphs [0315], [0316], and [0325]).
With regard to claim 92, which recites “the method of claim 90, further comprising delivering an mRNA encoding the prime editor into the cell,” as set forth above, Fraser and Komor collectively disclose the instantly claimed prime editor. While Fraser discloses that the reverse transcriptase and Cas effector taught therein may be expressed from a vector or plasmid (paragraphs [0026], [0027], and [0145]), Fraser does not explicitly disclose that these components are delivered as an mRNA. This deficiency is cured by Zhang, which discloses that the editing compositions disclosed therein (including Cas-enzyme fusions) may be delivered as mRNA molecules encoding all of the components thereof (paragraphs [0318] and [0319]).
With regard to claim 93, which recites “the mRNA and the pegRNA [of the method of claim 92] are delivered via a lipid nanoparticle,” Zhang further discloses that the mRNA encoding the components of the system taught therein (and set forth above) may be delivered via one or more lipid nanoparticles (paragraphs [0318] and [0319]).
With regard to claim 94, which recites “the method of claim 1, further comprising contacting the double stranded DNA with a complex comprising the prime editor and a nicking guide RNA (ngRNA),” Fraser further discloses that providing at least two DNA-targeting RNAs targeting opposite DNA strands facilitates a double-nicked double-strand break that favors HDR and decreases the frequency of insertion/deletion mutations at off-target DNA sites (paragraph [0143]). Per Fraser, two nickases of the invention disclosed therein may be utilized to create two single-stranded breaks on opposite strands of a target DNA, as set forth above (paragraph [0066]).
With regard to claim 95, which recites “the ngRNA [of the method of claim 94] comprises a[n] ngRNA spacer sequence capable of targeting the napDNAbp to the non-target strand of the double-stranded DNA sequence,” as set forth above, Fraser further discloses that providing at least two DNA-targeting RNAs targeting opposite DNA strands facilitates a double-nicked double-strand break that favors HDR and decreases the frequency of insertion/deletion mutations at off-target DNA sites (paragraph [0143]). Per Fraser, two nickases of the invention disclosed therein may be utilized to create two single-stranded breaks on opposite strands of a target DNA (paragraph [0066]). The DNA-targeting RNAs of Fraser read on the guide RNAs of Fraser, which are disclosed to comprise a crRNA spacer sequence that is complementary to a target nucleic acid sequence (paragraph [0127]).
Given that patent ‘770 discloses identical editing machinery, that Huang and UniProt disclose the napDNAbp and MMLV-RT sequences of the instant and patented applications, that Fraser and Komor collectively disclose the prime editing method claimed herein and modification thereof, and that Zhang discloses the utility of including an MS2 coat protein and associated binding aptamer thereof in such systems, 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 methods of patent ‘770 to utilize the disclosed sequences in place of the generic proteins claimed therein, as well as to include an MS2 coat protein and associated binding aptamer thereof to predictably prime edit a double-stranded DNA, as instantly claimed. One would have been motivated to make such a modification in order to receive the expected benefit of practicing prime editing.
Claims 1, 11, 13-16, 82-90, and 92-95 are rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1 of U.S. Patent No. 11,643,652 B2 (as cited in the IDS filed 08/24/2023) in view of WO 2018/049168 A1 (hereinafter Fraser; as cited in the IDS filed 08/24/2023), Komor et al., 2017 (hereinafter Komor; as cited in the IDS filed 08/24/2023), US 2016/0289673 A1 (hereinafter Huang), Ohtsubo et al., 2017 (hereinafter Ohtsubo), Yin et al., 2018 (hereinafter Yin), UniProt Entry P03355 (hereinafter UniProt; first entered 07/21/1986), and WO 2019/005884 (hereinafter Zhang; as cited in the IDS filed 08/24/2023).
MPEP 804 II B 1 states:
The specification can be used as a dictionary to learn the meaning of a term in the patent claim. Toro Co. v. White Consol. Indus., Inc., 199 F.3d 1295, 1299, 53 USPQ2d 1065, 1067 (Fed. Cir. 1999)… Further, those portions of the specification which provide support for the patent claims may also be examined and considered when addressing the issue of whether a claim in the application defines an obvious variation of an invention claimed in the patent. In re Vogel, 422 F.2d 438, 441-42, 164 USPQ 619, 622 (CCPA 1970).
The following rejections are in view of the decision of the Court of Appeals for the Federal Circuit in Pfizer Inc, v Teva pharmaceuticals USA Inc., 86 USPQ2d 1001, at page 1008 (March 2008), which indicates that there is no patentable distinction between claims to a product and a method of using that product disclosed in the specification of the application and that the preclusion of such a double patenting rejection under 35 USC 121 does not apply where the present application is other than a divisional application of the patent application containing such patentably indistinct claims.
Claim 1 of patent ‘652 is drawn to a prime editing guide RNA (PEgRNA), wherein said PEgRNA comprises a spacer sequence, an RNA extension arm that comprises a DNA synthesis template and a primer binding site, and a gRNA core that associates with a nucleic acid programmable DNA binding protein (napDNAbp) having nicking activity (i.e. a Cas9), wherein the primer binding site comprises a region of complementarity to a region upstream of a nick site in a second strand of the double-stranded target DNA sequence, and wherein the DNA synthesis template comprises a region of complementarity to a region downstream of the nick site in the second strand of the double-stranded target DNA sequence and comprises one or more nucleotide changes compared to the double-stranded target DNA sequence.
In comparison, instant claim 1 is also drawn to a PEgRNA comprising a spacer sequence capable of targeting the napDNAbp to a target strand of the double stranded DNA sequence, a guide RNA core capable of binding to the napDNAbp, and a DNA extension arm comprising a DNA synthesis template encoding the desired nucleotide change and a primer binding site capable of hybridizing to the single stranded DNA having the 3’ end. The napDNAbp of the instant application is limited to a Cas9 nickase at instant claim 11.
Regarding the additional limitations of the instant claim set, as set forth above, the prior art discloses these limitations.
With regard to claim 1, which recites “a method for editing a double-stranded DNA sequence, the method comprising: contacting the double-stranded DNA sequence with a complex comprising a prime editor and a prime editing guide RNA (PEgRNA), wherein the prime editor comprises (i) a nucleic acid programmable DNA binding protein (napDNAbp) capable of nicking a non-target strand of the double stranded DNA sequence to produce a single strand[ed] DNA having a 3’ end and (ii) a polymerase, and wherein the PEgRNA comprises: (i) a spacer sequence capable of targeting the napDNAbp to a target strand of the double stranded DNA sequence, (ii) a gRNA core capable of binding to the napDNAbp, and (iii) a DNA extension arm comprising a DNA synthesis template encoding the desired nucleotide change and a primer binding site capable of hybridizing to the single strand[ed] DNA having the 3’ end,” Fraser discloses methods for high-efficiency genome editing, wherein the method comprises providing retron-guide RNA cassettes (abstract). The retron-guide RNA cassettes of Fraser read on the instantly claimed PEgRNA, as they comprise a retron transcript (i.e. the extension arm of the claim) and a guide RNA molecule comprising a spacer complementary to a first strand and a guide RNA core capable of binding to the Cas napDNAbp taught therein (paragraphs [0003], [0006], [0009], [0010], and [0127]). As set forth above, patent ‘652 also recites PEgRNAs. Fraser explicitly discloses that the retron transcript of the retron-guide RNA cassettes taught therein is capable of self-priming reverse transcription by a reverse transcriptase (paragraph [0020]). The reverse transcriptase of Fraser (see paragraphs [0106], [0147], [0148], and [0154]) reads on the instantly claimed polymerase (see paragraph [0010] of the instant specification), while the Cas nickase disclosed therein (see paragraphs [0066], [0138], [0142], and [0143]) read on the instantly claimed napDNAbp capable of nicking a strand of the double stranded DNA sequence to produce a single stranded DNA having a 3’ end. Thus, Fraser discloses each and every limitation of the instantly claimed method with the exception of specifically nicking the non-target strand. This deficiency is cured by Komor.
While the disclosure of Fraser is drawn to coordinated actions of a Cas nickase (paragraphs [0066], [0138], [0142], and [0143]) and a reverse transcriptase (paragraphs [0020], [0106], [0147], [0148], and [0154]), Cas nickase activity has been coordinated with other effector proteins. Komor discloses base editors, which comprise a nickase Cas9 such as nSpCas9 and nSaCas9 fused to a base editing enzyme such as APOBEC1, wherein the Cas9 nickase nicks the non-edited strand in order to facilitate base editing of the targeted residue in the edited (i.e. targeted) strand (Figure 2). While the disclosure of Fraser and Komor differ in their effects, both disclosures are drawn to coordinated actions of a Cas nickase and an effector protein for purposes of editing a targeted nucleic acid. Therefore, based on these disclosures, one of ordinary skill in the art would have been motivated, prior to the effective filing date of the instant application, to apply the principles disclosed in Komor to the methods of Fraser such that the Cas nickase disclosed therein specifically nicks the non-targeted strand in order to specifically edit the targeted strand, as set forth in greater detail below.
With regard to claim 11, which recites “the napDNAbp [of the method of claim 1] is a Cas9 nickase (nCas9),” as set forth above, Fraser discloses coordinated actions of a Cas nickase and a reverse transcriptase, wherein the Cas nickase is specifically a Cas9 nickase (paragraph [0143]).
With regard to claim 13, which recites “the napDNAbp [of the method of claim 1] comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 137,” as set forth above, Fraser discloses that a Cas9 nickase may be used in the editing methods disclosed therein. However, Fraser is silent as to the sequence of said Cas9 nickase. This deficiency is cured by Huang.
Huang discloses that an exemplary nicking Cas9 protein is Cas9 H840A, which corresponds to SEQ ID NO: 6 taught therein (paragraphs [0077] and [0080]). As shown in the alignment of the Appendix, SEQ ID NO: 6 of Huang comprises 100% identity to instant SEQ ID NO: 137.
With regard to claim 14, which recites “the polymerase [of the method of claim 1] is a reverse transcriptase,” as set forth above, Fraser discloses coordinated actions of a Cas nickase and a reverse transcriptase (paragraphs [0009], [0106], [0147], [0148], and [0154]).
With regard to claim 15, which recites “the reverse transcriptase [of the method of claim 14] comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 139,” as set forth above, Fraser discloses that a reverse transcriptase may be used in the editing methods disclosed therein. However, Fraser is silent as to the sequence of said reverse transcriptase. This deficiency is cured by Ohtsubo, Yin, and Uniprot, as set forth below.
Ohtsubo teaches that the Moloney murine leukemia virus (MMLV) reverse transcriptase (MMLV-RT) is widely used for cDNA synthesis (i.e. RNA to DNA) but also has DNA polymerase activity when using DNA as a template (page 1, paragraph 3 of the introduction). Per Yin, adding DNA residues to CRISPR guide RNAs significantly reduces off-target genome editing and reduces the associated cost (abstract). Therefore, prior to the effective filing date of the instant invention, one of ordinary skill in the art would have been motivated to generate a guide RNA per the teachings of Yin that comprises DNA residues. Therefore, the reverse transcriptase disclosed in Fraser must be capable of synthesizing DNA from both DNA and RNA in order to utilize both chimeric guide RNAs and standard guide RNAs efficiently. As set forth above, Ohtsubo teaches that MMLV-RT is uniquely suited to both RNA to DNA synthesis and DNA to DNA synthesis. Accordingly, one of ordinary skill in the art would have been motivated to utilize MMLV-RT in the methods of Fraser. UniProt teaches the sequence of MMLV-RT, which comprises 98.8% sequence identity to instant SEQ ID NO: 139, as shown in the alignment of the Appendix.
With regard to claim 16, which recites “the DNA extension arm [of the method of claim 1] is at the 3’ end of the PEgRNA,” Fraser further discloses that the retron-guide RNA cassette taught therein may be structured such that the gRNA coding region is located 3’ or 5’ of the retron (paragraphs [0010] and [0102]).
With regard to claim 82, which recites “the prime editor [of the method of claim 1] is a fusion protein,” as set forth above, Fraser discloses coordinated actions of a Cas nickase and a reverse transcriptase for purposes of editing a nucleic acid sequence. However, Fraser does not explicitly disclose fusion of the Cas nickase and the reverse transcriptase taught therein. This deficiency is cured by Komor. Komor discloses base editors, which comprise a nickase Cas9 such as nSpCas9 and nSaCas9 fused to a base editing enzyme such as APOBEC1 (Figure 2). While the disclosure of Fraser and Komor differ in their effects, both disclosures are drawn to coordinated actions of a Cas nickase and an effector protein for purposes of editing a targeted nucleic acid. Therefore, based on these disclosures, one of ordinary skill in the art would have been motivated, prior to the effective filing date of the instant application, to apply the principles disclosed in Komor to the methods of Fraser such that the Cas nickase disclosed therein is fused to the reverse transcriptase disclosed therein.
With regard to claim 83, which recites “the napDNAbp [of the method of claim 82] is N-terminal of the polymerase in the fusion protein,” as set forth above, Fraser and Komor collectively disclose a fusion protein comprising a Cas nickase and a reverse transcriptase. However, neither specifically teaches the architecture of said fusion protein. Given that the fusion protein collectively disclosed by Fraser and Komor comprises only two protein components, there are only two possible arrangements: one in which the Cas nickase is N-terminal of the reverse transcriptase in the fusion protein, and one in which the Cas nickase is C-terminal of the reverse transcriptase in the fusion protein. Per MPEP § 2144.04(VI)(C), it is not inventive to rearrange parts. Given that there are only a small, finite number of possible arrangements, it is not considered inventive to merely rearrange these components.
With regard to claim 84, which recites “the prime editor [of the method of claim 83] further comprises a peptide linker between the napDNAbp and the polymerase,” as set forth above, Fraser and Komor collectively disclose the method of claim 83. However, neither Fraser nor Komor disclose the peptide linker of instant claim 84. This deficiency is cured by Zhang.
Zhang discloses methods and compositions for targeting and editing nucleic acids, particularly RNA targeted and edited via a Cas13 fusion protein comprising a Cas13 fused to an editing enzyme such as ADAR (abstract; paragraph [0047]; Figure 2). While the methods of Zhang are drawn to RNA editing and not DNA editing, Zhang nonetheless discloses coordinated activity of a Cas enzyme and an editing enzyme, as in the instant application, Fraser, and Komor. Zhang further discloses that the fusion proteins taught therein may be linked via a peptide linker (paragraphs [0200] and [0201]). Thus, Zhang establishes that Cas proteins may be fused to effector proteins via a peptide linker, as instantly claimed.
With regard to claim 85, which recites “the polymerase [of the method of claim 1] is not connected to the napDNAbp in a fusion protein,” as set forth above, Fraser discloses coordinated actions of a Cas9 nickase and a reverse transcriptase for purposes of editing a nucleic acid sequence. Fraser further discloses that the reverse transcriptase and Cas effector disclosed therein may be provided separately (paragraphs [0026] and [0027]).
With regard to claim 86, which recites “the PEgRNA further comprises an RNA-binding protein recruitment domain,” as set forth above, the retron-guide cassette of Fraser reads on the instantly claimed PEgRNA. Zhang further discloses that the guide molecule taught therein comprises an aptamer sequence capable of binding to an RNA-binding adaptor protein such as MS2 (paragraphs [0011], [0315], and [0316), which reads on the instantly claimed RNA-binding protein recruitment domain.
With regard to claim 87, which recites “the RNA-binding protein recruitment domain [of the method of claim 86] is an MS2 aptamer,” as set forth above, Zhang further discloses that the guide molecule taught therein comprises an aptamer sequence capable of binding to an RNA-binding adaptor protein such as MS2 (paragraphs [0011], [0315], and [0316).
With regard to claim 88, which recites “the polymerase [of the method of claim 85] is fused to an RNA-binding protein domain,” Zhang further discloses that the adenosine deaminase effector taught therein may be fused to RNA-binding protein MS2 coat protein (paragraphs [0315] and [0316]). While the adenosine deaminase effector of Zhang does not read on the instantly claimed polymerase, the disclosure of Zhang is applied to establish that principles of coordinating activity of a Cas protein and an editing enzyme, as in the instant application, Fraser, and Komor. Therefore, Zhang teaches that the effector/editing enzyme taught therein may be fused to an RNA-binding protein such as MS2 coat protein for purposes of implementing orthogonal gene editing (paragraphs [0315], [0316], and [0325]).
With regard to claim 89, which recites “the polymerase [of the method of claim 86] is fused to an MS2cp domain,” as set forth above, Zhang further discloses that the adenosine deaminase effector taught therein may be fused to RNA-binding protein MS2 coat protein for purposes of implementing orthogonal gene editing (paragraphs [0315], [0316], and [0325]).
With regard to claim 90, which recites “the double stranded DNA [of the method of claim 1] is in a cell,” Fraser discloses that the methods taught therein may be practiced within a genome of a host cell, wherein the host cell is transformed with a vector carrying the machinery set forth above and is subsequently edited (paragraph [0024]).
With regard to claim 92, which recites “the method of claim 90, further comprising delivering an mRNA encoding the prime editor into the cell,” as set forth above, Fraser and Komor collectively disclose the instantly claimed prime editor. While Fraser discloses that the reverse transcriptase and Cas effector taught therein may be expressed from a vector or plasmid (paragraphs [0026], [0027], and [0145]), Fraser does not explicitly disclose that these components are delivered as an mRNA. This deficiency is cured by Zhang, which discloses that the editing compositions disclosed therein (including Cas-enzyme fusions) may be delivered as mRNA molecules encoding all of the components thereof (paragraphs [0318] and [0319]).
With regard to claim 93, which recites “the mRNA and the pegRNA [of the method of claim 92] are delivered via a lipid nanoparticle,” Zhang further discloses that the mRNA encoding the components of the system taught therein (and set forth above) may be delivered via one or more lipid nanoparticles (paragraphs [0318] and [0319]).
With regard to claim 94, which recites “the method of claim 1, further comprising contacting the double stranded DNA with a complex comprising the prime editor and a nicking guide RNA (ngRNA),” Fraser further discloses that providing at least two DNA-targeting RNAs targeting opposite DNA strands facilitates a double-nicked double-strand break that favors HDR and decreases the frequency of insertion/deletion mutations at off-target DNA sites (paragraph [0143]). Per Fraser, two nickases of the invention disclosed therein may be utilized to create two single-stranded breaks on opposite strands of a target DNA, as set forth above (paragraph [0066]).
With regard to claim 95, which recites “the ngRNA [of the method of claim 94] comprises a[n] ngRNA spacer sequence capable of targeting the napDNAbp to the non-target strand of the double-stranded DNA sequence,” as set forth above, Fraser further discloses that providing at least two DNA-targeting RNAs targeting opposite DNA strands facilitates a double-nicked double-strand break that favors HDR and decreases the frequency of insertion/deletion mutations at off-target DNA sites (paragraph [0143]). Per Fraser, two nickases of the invention disclosed therein may be utilized to create two single-stranded breaks on opposite strands of a target DNA (paragraph [0066]). The DNA-targeting RNAs of Fraser read on the guide RNAs of Fraser, which are disclosed to comprise a crRNA spacer sequence that is complementary to a target nucleic acid sequence (paragraph [0127]).
Given that patent ‘652 also discloses PEgRNAs, as in the instant application, that Fraser and Komor collectively disclose a method of editing a target genome utilizing said PEgRNAs, that Huang and UniProt disclose the napDNAbp and MMLV-RT sequences of the instant and patented applications, and that Zhang discloses the utility of including an MS2 coat protein and associated binding aptamer thereof in such systems, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to utilize the PEgRNA of patent ‘652 in the systems collectively disclosed by the prior art, as well as to include an MS2 coat protein and associated binding aptamer thereof to predictably prime edit a double-stranded DNA, as instantly claimed. One would have been motivated to make such a modification in order to receive the expected benefit of practicing prime editing.
Claims 1, 11, 13-16, 82-90, and 92-95 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 10, 12, 15, 16, 22, 24, and 30 of U.S. Patent No. 11,795,452 B2 (as cited in the IDS filed 07/15/2026) in view of WO 2018/049168 A1 (hereinafter Fraser; as cited in the IDS filed 08/24/2023), Komor et al., 2017 (hereinafter Komor; as cited in the IDS filed 08/24/2023), US 2016/0289673 A1 (hereinafter Huang), Ohtsubo et al., 2017 (hereinafter Ohtsubo), Yin et al., 2018 (hereinafter Yin), UniProt Entry P03355 (hereinafter UniProt; first entered 07/21/1986), and WO 2019/005884 (hereinafter Zhang; as cited in the IDS filed 08/24/2023).
MPEP 804 II B 1 states:
The specification can be used as a dictionary to learn the meaning of a term in the patent claim. Toro Co. v. White Consol. Indus., Inc., 199 F.3d 1295, 1299, 53 USPQ2d 1065, 1067 (Fed. Cir. 1999)… Further, those portions of the specification which provide support for the patent claims may also be examined and considered when addressing the issue of whether a claim in the application defines an obvious variation of an invention claimed in the patent. In re Vogel, 422 F.2d 438, 441-42, 164 USPQ 619, 622 (CCPA 1970).
The following rejections are in view of the decision of the Court of Appeals for the Federal Circuit in Pfizer Inc, v Teva pharmaceuticals USA Inc., 86 USPQ2d 1001, at page 1008 (March 2008), which indicates that there is no patentable distinction between claims to a product and a method of using that product disclosed in the specification of the application and that the preclusion of such a double patenting rejection under 35 USC 121 does not apply where the present application is other than a divisional application of the patent application containing such patentably indistinct claims.
Claim 1 of patent ‘452 is drawn to a prime editing system comprising a PEgRNA comprising a spacer sequence, an extension arm comprising a DNA synthesis template and a primer binding site, and a guide RNA core that interacts with a napDNAbp, and a prime editor comprising said napDNAbp (wherein said napDNAbp is a Cas9 nickase) and a reverse transcriptase (also recited at patented claims 15 and 16). The napDNAbp is limited to a Cas9 nickase at patented claim 12.
This system is identical to that utilized in the recitation of the method of instant claims 1, 11, and 14. Regarding the method of using said system, as set forth above, Fraser discloses methods for high-efficiency genome editing, wherein the method comprises providing retron-guide RNA cassettes (abstract). The retron-guide RNA cassettes of Fraser read on the instantly claimed PEgRNA, as they comprise a retron transcript (i.e. the extension arm of the claim) and a guide RNA molecule comprising a spacer complementary to a first strand and a guide RNA core capable of binding to the Cas napDNAbp taught therein (paragraphs [0003], [0006], [0009], [0010], and [0127]).
Claim 10 of patent ‘452 further recites that the system claimed therein further comprises a nicking single guide RNA, wherein the nicking sgRNA comprises a spacer sequence comprising a region of complementarity to the second strand of the double-stranded target DNA.
This system is identical to that utilized in the recitation of the method of instant claims 94 and 95.
Finally, claim 22 of patent ‘452 further recites that the napDNAbp and the reverse transcriptase claimed therein are connected to form a fusion protein, as recited at instant claim 82.
Regarding the additional limitations of the instant claim set, as set forth above, the prior art discloses these limitations.
With regard to claim 13, which recites “the napDNAbp [of the method of claim 1] comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 137,” as set forth above, Fraser discloses that a Cas9 nickase may be used in the editing methods disclosed therein. However, Fraser is silent as to the sequence of said Cas9 nickase. This deficiency is cured by Huang.
Huang discloses that an exemplary nicking Cas9 protein is Cas9 H840A, which corresponds to SEQ ID NO: 6 taught therein (paragraphs [0077] and [0080]). As shown in the alignment of the Appendix, SEQ ID NO: 6 of Huang comprises 100% identity to instant SEQ ID NO: 137.
The Examiner notes that the patented application also recites that the Cas9 nickase claimed therein comprises an H840A amino acid substitution at patented claim 30.
With regard to claim 15, which recites “the reverse transcriptase [of the method of claim 14] comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 139,” as set forth above, Fraser discloses that a reverse transcriptase may be used in the editing methods disclosed therein. However, Fraser is silent as to the sequence of said reverse transcriptase. This deficiency is cured by Ohtsubo, Yin, and Uniprot, as set forth below.
Ohtsubo teaches that the Moloney murine leukemia virus (MMLV) reverse transcriptase (MMLV-RT) is widely used for cDNA synthesis (i.e. RNA to DNA) but also has DNA polymerase activity when using DNA as a template (page 1, paragraph 3 of the introduction). Per Yin, adding DNA residues to CRISPR guide RNAs significantly reduces off-target genome editing and reduces the associated cost (abstract). Therefore, prior to the effective filing date of the instant invention, one of ordinary skill in the art would have been motivated to generate a guide RNA per the teachings of Yin that comprises DNA residues. Therefore, the reverse transcriptase disclosed in Fraser must be capable of synthesizing DNA from both DNA and RNA in order to utilize both chimeric guide RNAs and standard guide RNAs efficiently. As set forth above, Ohtsubo teaches that MMLV-RT is uniquely suited to both RNA to DNA synthesis and DNA to DNA synthesis. Accordingly, one of ordinary skill in the art would have been motivated to utilize MMLV-RT in the methods of Fraser. UniProt teaches the sequence of MMLV-RT, which comprises 98.8% sequence identity to instant SEQ ID NO: 139, as shown in the alignment of the Appendix.
The Examiner notes that the patented application also recites that the reverse transcriptase claimed therein is an MMLV-RT at patented claim 24.
With regard to claim 16, which recites “the DNA extension arm [of the method of claim 1] is at the 3’ end of the PEgRNA,” Fraser further discloses that the retron-guide RNA cassette taught therein may be structured such that the gRNA coding region is located 3’ or 5’ of the retron (paragraphs [0010] and [0102]).
With regard to claim 83, which recites “the napDNAbp [of the method of claim 82] is N-terminal of the polymerase in the fusion protein,” as set forth above, Fraser and Komor collectively disclose a fusion protein comprising a Cas nickase and a reverse transcriptase. However, neither specifically teaches the architecture of said fusion protein. Given that the fusion protein collectively disclosed by Fraser and Komor comprises only two protein components, there are only two possible arrangements: one in which the Cas nickase is N-terminal of the reverse transcriptase in the fusion protein, and one in which the Cas nickase is C-terminal of the reverse transcriptase in the fusion protein. Per MPEP § 2144.04(VI)(C), it is not inventive to rearrange parts. Given that there are only a small, finite number of possible arrangements, it is not considered inventive to merely rearrange these components.
With regard to claim 84, which recites “the prime editor [of the method of claim 83] further comprises a peptide linker between the napDNAbp and the polymerase,” as set forth above, Fraser and Komor collectively disclose the method of claim 83. However, neither Fraser nor Komor disclose the peptide linker of instant claim 84. This deficiency is cured by Zhang.
Zhang discloses methods and compositions for targeting and editing nucleic acids, particularly RNA targeted and edited via a Cas13 fusion protein comprising a Cas13 fused to an editing enzyme such as ADAR (abstract; paragraph [0047]; Figure 2). While the methods of Zhang are drawn to RNA editing and not DNA editing, Zhang nonetheless discloses coordinated activity of a Cas enzyme and an editing enzyme, as in the instant application, Fraser, and Komor. Zhang further discloses that the fusion proteins taught therein may be linked via a peptide linker (paragraphs [0200] and [0201]). Thus, Zhang establishes that Cas proteins may be fused to effector proteins via a peptide linker, as instantly claimed.
With regard to claim 85, which recites “the polymerase [of the method of claim 1] is not connected to the napDNAbp in a fusion protein,” as set forth above, Fraser discloses coordinated actions of a Cas9 nickase and a reverse transcriptase for purposes of editing a nucleic acid sequence. Fraser further discloses that the reverse transcriptase and Cas effector disclosed therein may be provided separately (paragraphs [0026] and [0027]).
With regard to claim 86, which recites “the PEgRNA further comprises an RNA-binding protein recruitment domain,” as set forth above, the retron-guide cassette of Fraser reads on the instantly claimed PEgRNA. Zhang further discloses that the guide molecule taught therein comprises an aptamer sequence capable of binding to an RNA-binding adaptor protein such as MS2 (paragraphs [0011], [0315], and [0316), which reads on the instantly claimed RNA-binding protein recruitment domain.
With regard to claim 87, which recites “the RNA-binding protein recruitment domain [of the method of claim 86] is an MS2 aptamer,” as set forth above, Zhang further discloses that the guide molecule taught therein comprises an aptamer sequence capable of binding to an RNA-binding adaptor protein such as MS2 (paragraphs [0011], [0315], and [0316).
With regard to claim 88, which recites “the polymerase [of the method of claim 85] is fused to an RNA-binding protein domain,” Zhang further discloses that the adenosine deaminase effector taught therein may be fused to RNA-binding protein MS2 coat protein (paragraphs [0315] and [0316]). While the adenosine deaminase effector of Zhang does not read on the instantly claimed polymerase, the disclosure of Zhang is applied to establish that principles of coordinating activity of a Cas protein and an editing enzyme, as in the instant application, Fraser, and Komor. Therefore, Zhang teaches that the effector/editing enzyme taught therein may be fused to an RNA-binding protein such as MS2 coat protein for purposes of implementing orthogonal gene editing (paragraphs [0315], [0316], and [0325]).
With regard to claim 89, which recites “the polymerase [of the method of claim 86] is fused to an MS2cp domain,” as set forth above, Zhang further discloses that the adenosine deaminase effector taught therein may be fused to RNA-binding protein MS2 coat protein for purposes of implementing orthogonal gene editing (paragraphs [0315], [0316], and [0325]).
With regard to claim 90, which recites “the double stranded DNA [of the method of claim 1] is in a cell,” Fraser discloses that the methods taught therein may be practiced within a genome of a host cell, wherein the host cell is transformed with a vector carrying the machinery set forth above and is subsequently edited (paragraph [0024]).
With regard to claim 92, which recites “the method of claim 90, further comprising delivering an mRNA encoding the prime editor into the cell,” as set forth above, Fraser and Komor collectively disclose the instantly claimed prime editor. While Fraser discloses that the reverse transcriptase and Cas effector taught therein may be expressed from a vector or plasmid (paragraphs [0026], [0027], and [0145]), Fraser does not explicitly disclose that these components are delivered as an mRNA. This deficiency is cured by Zhang, which discloses that the editing compositions disclosed therein (including Cas-enzyme fusions) may be delivered as mRNA molecules encoding all of the components thereof (paragraphs [0318] and [0319]).
With regard to claim 93, which recites “the mRNA and the pegRNA [of the method of claim 92] are delivered via a lipid nanoparticle,” Zhang further discloses that the mRNA encoding the components of the system taught therein (and set forth above) may be delivered via one or more lipid nanoparticles (paragraphs [0318] and [0319]).
Given that patent ‘452 also discloses PEgRNAs as part of a prime editing composition further comprising a napDNAbp and a reverse transcriptase, as in the instant application, that Fraser and Komor collectively disclose a method of editing a target genome utilizing said PEgRNAs, that Huang and UniProt disclose the napDNAbp and MMLV-RT sequences of the instant and patented applications, and that Zhang discloses the utility of including an MS2 coat protein and associated binding aptamer thereof in such systems, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to utilize the composition of patent ‘452 in the systems collectively disclosed by the prior art, as well as to include an MS2 coat protein and associated binding aptamer thereof to predictably prime edit a double-stranded DNA, as instantly claimed. One would have been motivated to make such a modification in order to receive the expected benefit of practicing prime editing.
Claims 1, 11, 13-16, 82-90, and 92-95 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1 and 21-31 of U.S. Patent No. 12,624,354 B2 (as cited in the IDS filed 07/15/2026) in view of WO 2018/049168 A1 (hereinafter Fraser; as cited in the IDS filed 08/24/2023), Komor et al., 2017 (hereinafter Komor; as cited in the IDS filed 08/24/2023), US 2016/0289673 A1 (hereinafter Huang), Ohtsubo et al., 2017 (hereinafter Ohtsubo), Yin et al., 2018 (hereinafter Yin), UniProt Entry P03355 (hereinafter UniProt; first entered 07/21/1986), and WO 2019/005884 (hereinafter Zhang; as cited in the IDS filed 08/24/2023).
MPEP 804 II B 1 states:
The specification can be used as a dictionary to learn the meaning of a term in the patent claim. Toro Co. v. White Consol. Indus., Inc., 199 F.3d 1295, 1299, 53 USPQ2d 1065, 1067 (Fed. Cir. 1999)… Further, those portions of the specification which provide support for the patent claims may also be examined and considered when addressing the issue of whether a claim in the application defines an obvious variation of an invention claimed in the patent. In re Vogel, 422 F.2d 438, 441-42, 164 USPQ 619, 622 (CCPA 1970).
The following rejections are in view of the decision of the Court of Appeals for the Federal Circuit in Pfizer Inc, v Teva pharmaceuticals USA Inc., 86 USPQ2d 1001, at page 1008 (March 2008), which indicates that there is no patentable distinction between claims to a product and a method of using that product disclosed in the specification of the application and that the preclusion of such a double patenting rejection under 35 USC 121 does not apply where the present application is other than a divisional application of the patent application containing such patentably indistinct claims.
Patented claim 1 recites “a prime editing system,” wherein said system comprises a PEgRNA comprising a spacer sequence, a gRNA core, and an RNA extension arm comprising a DNA synthesis template and a primer binding site, as well as a prime editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase domain. The napDNAbp of patent ‘354 is recited to be a CRISPR-Cas effector protein comprising a RuvC nuclease domain and/or an HNH nuclease domain that is a nickase per patented claims 22-25. The reverse transcriptase of patent ‘354 is recited to be from a retrovirus or a retrotransposon (i.e. an M-MLV RT) at patented claims 26 and 27.
The system of patent ‘354 reads on the system of the instantly claimed methods. Regarding the method of using said system, as set forth above, Fraser discloses methods for high-efficiency genome editing, wherein the method comprises providing retron-guide RNA cassettes (abstract). The retron-guide RNA cassettes of Fraser read on the instantly claimed PEgRNA, as they comprise a retron transcript (i.e. the extension arm of the claim) and a guide RNA molecule comprising a spacer complementary to a first strand and a guide RNA core capable of binding to the Cas napDNAbp taught therein (paragraphs [0003], [0006], [0009], [0010], and [0127]).
Regarding the patented napDNAbp limitations, these recitations are satisfied by instant claim 11. Regarding the patented reverse transcriptase limitations, these recitations are satisfied by instant claim 14. The Examiner notes that while the instant application is drawn in part to a Cas9 H840A nickase and an M-MLV reverse transcriptase, the instant claim set requires sequences not recited in the patented application. Additionally, while patented claim 31 does recite mutation(s) to the HNH nuclease domain that decrease or eliminate nuclease activity, this recitation does not comprise the sequence of the instant application. However, the art teaches these sequences, as set forth below.
With regard to claim 13, which recites “the napDNAbp [of the method of claim 1] comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 137,” as set forth above, Fraser discloses that a Cas9 nickase may be used in the editing methods disclosed therein. However, Fraser is silent as to the sequence of said Cas9 nickase. This deficiency is cured by Huang.
Huang discloses that an exemplary nicking Cas9 protein is Cas9 H840A, which corresponds to SEQ ID NO: 6 taught therein (paragraphs [0077] and [0080]). As shown in the alignment of the Appendix, SEQ ID NO: 6 of Huang comprises 100% identity to instant SEQ ID NO: 137.
With regard to claim 15, which recites “the reverse transcriptase [of the method of claim 14] comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 139,” as set forth above, Fraser discloses that a reverse transcriptase may be used in the editing methods disclosed therein. However, Fraser is silent as to the sequence of said reverse transcriptase. This deficiency is cured by Ohtsubo, Yin, and Uniprot, as set forth below.
Ohtsubo teaches that the Moloney murine leukemia virus (MMLV) reverse transcriptase (MMLV-RT) is widely used for cDNA synthesis (i.e. RNA to DNA) but also has DNA polymerase activity when using DNA as a template (page 1, paragraph 3 of the introduction). Per Yin, adding DNA residues to CRISPR guide RNAs significantly reduces off-target genome editing and reduces the associated cost (abstract). Therefore, prior to the effective filing date of the instant invention, one of ordinary skill in the art would have been motivated to generate a guide RNA per the teachings of Yin that comprises DNA residues. Therefore, the reverse transcriptase disclosed in Fraser must be capable of synthesizing DNA from both DNA and RNA in order to utilize both chimeric guide RNAs and standard guide RNAs efficiently. As set forth above, Ohtsubo teaches that MMLV-RT is uniquely suited to both RNA to DNA synthesis and DNA to DNA synthesis. Accordingly, one of ordinary skill in the art would have been motivated to utilize MMLV-RT in the methods of Fraser. UniProt teaches the sequence of MMLV-RT, which comprises 98.8% sequence identity to instant SEQ ID NO: 139, as shown in the alignment of the Appendix.
With regard to claim 16, which recites “the DNA extension arm [of the method of claim 1] is at the 3’ end of the PEgRNA,” Fraser further discloses that the retron-guide RNA cassette taught therein may be structured such that the gRNA coding region is located 3’ or 5’ of the retron (paragraphs [0010] and [0102]).
Patented claim 28 further recites that the napDNAbp and the reverse transcriptase are connected as a fusion protein, as recited at instant claim 82.
With regard to claim 83, which recites “the napDNAbp [of the method of claim 82] is N-terminal of the polymerase in the fusion protein,” as set forth above, Fraser and Komor collectively disclose a fusion protein comprising a Cas nickase and a reverse transcriptase. However, neither specifically teaches the architecture of said fusion protein. Given that the fusion protein collectively disclosed by Fraser and Komor comprises only two protein components, there are only two possible arrangements: one in which the Cas nickase is N-terminal of the reverse transcriptase in the fusion protein, and one in which the Cas nickase is C-terminal of the reverse transcriptase in the fusion protein. Per MPEP § 2144.04(VI)(C), it is not inventive to rearrange parts. Given that there are only a small, finite number of possible arrangements, it is not considered inventive to merely rearrange these components.
With regard to claim 84, which recites “the prime editor [of the method of claim 83] further comprises a peptide linker between the napDNAbp and the polymerase,” as set forth above, Fraser and Komor collectively disclose the method of claim 83. However, neither Fraser nor Komor disclose the peptide linker of instant claim 84. This deficiency is cured by Zhang.
Zhang discloses methods and compositions for targeting and editing nucleic acids, particularly RNA targeted and edited via a Cas13 fusion protein comprising a Cas13 fused to an editing enzyme such as ADAR (abstract; paragraph [0047]; Figure 2). While the methods of Zhang are drawn to RNA editing and not DNA editing, Zhang nonetheless discloses coordinated activity of a Cas enzyme and an editing enzyme, as in the instant application, Fraser, and Komor. Zhang further discloses that the fusion proteins taught therein may be linked via a peptide linker (paragraphs [0200] and [0201]). Thus, Zhang establishes that Cas proteins may be fused to effector proteins via a peptide linker, as instantly claimed.
With regard to claim 85, which recites “the polymerase [of the method of claim 1] is not connected to the napDNAbp in a fusion protein,” as set forth above, Fraser discloses coordinated actions of a Cas9 nickase and a reverse transcriptase for purposes of editing a nucleic acid sequence. Fraser further discloses that the reverse transcriptase and Cas effector disclosed therein may be provided separately (paragraphs [0026] and [0027]).
With regard to claim 86, which recites “the PEgRNA further comprises an RNA-binding protein recruitment domain,” as set forth above, the retron-guide cassette of Fraser reads on the instantly claimed PEgRNA. Zhang further discloses that the guide molecule taught therein comprises an aptamer sequence capable of binding to an RNA-binding adaptor protein such as MS2 (paragraphs [0011], [0315], and [0316), which reads on the instantly claimed RNA-binding protein recruitment domain.
Additionally, patented claims 21 and 30 further recite the addition of an aptamer capable of recruiting an effector domain, wherein the aptamer is an MS2 aptamer, as recited at instant claim 87.
With regard to claim 88, which recites “the polymerase [of the method of claim 85] is fused to an RNA-binding protein domain,” Zhang further discloses that the adenosine deaminase effector taught therein may be fused to RNA-binding protein MS2 coat protein (paragraphs [0315] and [0316]). While the adenosine deaminase effector of Zhang does not read on the instantly claimed polymerase, the disclosure of Zhang is applied to establish that principles of coordinating activity of a Cas protein and an editing enzyme, as in the instant application, Fraser, and Komor. Therefore, Zhang teaches that the effector/editing enzyme taught therein may be fused to an RNA-binding protein such as MS2 coat protein for purposes of implementing orthogonal gene editing (paragraphs [0315], [0316], and [0325]).
With regard to claim 89, which recites “the polymerase [of the method of claim 86] is fused to an MS2cp domain,” as set forth above, Zhang further discloses that the adenosine deaminase effector taught therein may be fused to RNA-binding protein MS2 coat protein for purposes of implementing orthogonal gene editing (paragraphs [0315], [0316], and [0325]).
With regard to claim 90, which recites “the double stranded DNA [of the method of claim 1] is in a cell,” Fraser discloses that the methods taught therein may be practiced within a genome of a host cell, wherein the host cell is transformed with a vector carrying the machinery set forth above and is subsequently edited (paragraph [0024]).
With regard to claim 92, which recites “the method of claim 90, further comprising delivering an mRNA encoding the prime editor into the cell,” as set forth above, Fraser and Komor collectively disclose the instantly claimed prime editor. While Fraser discloses that the reverse transcriptase and Cas effector taught therein may be expressed from a vector or plasmid (paragraphs [0026], [0027], and [0145]), Fraser does not explicitly disclose that these components are delivered as an mRNA. This deficiency is cured by Zhang, which discloses that the editing compositions disclosed therein (including Cas-enzyme fusions) may be delivered as mRNA molecules encoding all of the components thereof (paragraphs [0318] and [0319]).
With regard to claim 93, which recites “the mRNA and the pegRNA [of the method of claim 92] are delivered via a lipid nanoparticle,” Zhang further discloses that the mRNA encoding the components of the system taught therein (and set forth above) may be delivered via one or more lipid nanoparticles (paragraphs [0318] and [0319]).
Patented claim 29 further recites that the system claimed therein further comprises a second-strand nicking guide RNA, which is also recited at instant claims 94 and 95.
Given that patent ‘354 also discloses PEgRNAs as part of a prime editing composition further comprising a napDNAbp and a reverse transcriptase, as in the instant application, that Fraser and Komor collectively disclose a method of editing a target genome utilizing said PEgRNAs, that Huang and UniProt disclose the napDNAbp and MMLV-RT sequences of the instant and patented applications, and that Zhang discloses the utility of including an MS2 coat protein and associated binding aptamer thereof in such systems, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to utilize the composition of patent ‘354 in the systems collectively disclosed by the prior art, as well as to include an MS2 coat protein and associated binding aptamer thereof to predictably prime edit a double-stranded DNA, as instantly claimed. One would have been motivated to make such a modification in order to receive the expected benefit of practicing prime editing.
Claims 1, 11, 13-16, 82-90, and 92-95 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1 and 26-30 of U.S. Patent No. 12,281,303 B2 (as cited in the IDS filed 07/15/2026) in view of WO 2018/049168 A1 (hereinafter Fraser; as cited in the IDS filed 08/24/2023), Komor et al., 2017 (hereinafter Komor; as cited in the IDS filed 08/24/2023), US 2016/0289673 A1 (hereinafter Huang), Ohtsubo et al., 2017 (hereinafter Ohtsubo), Yin et al., 2018 (hereinafter Yin), UniProt Entry P03355 (hereinafter UniProt; first entered 07/21/1986), and WO 2019/005884 (hereinafter Zhang; as cited in the IDS filed 08/24/2023).
MPEP 804 II B 1 states:
The specification can be used as a dictionary to learn the meaning of a term in the patent claim. Toro Co. v. White Consol. Indus., Inc., 199 F.3d 1295, 1299, 53 USPQ2d 1065, 1067 (Fed. Cir. 1999)… Further, those portions of the specification which provide support for the patent claims may also be examined and considered when addressing the issue of whether a claim in the application defines an obvious variation of an invention claimed in the patent. In re Vogel, 422 F.2d 438, 441-42, 164 USPQ 619, 622 (CCPA 1970).
The following rejections are in view of the decision of the Court of Appeals for the Federal Circuit in Pfizer Inc, v Teva pharmaceuticals USA Inc., 86 USPQ2d 1001, at page 1008 (March 2008), which indicates that there is no patentable distinction between claims to a product and a method of using that product disclosed in the specification of the application and that the preclusion of such a double patenting rejection under 35 USC 121 does not apply where the present application is other than a divisional application of the patent application containing such patentably indistinct claims.
Claim 1 of patent ‘303 is drawn to a prime editing guide RNA (PEgRNA), wherein said PEgRNA comprises a spacer sequence, an RNA extension arm that comprises a DNA synthesis template and a primer binding site, and a gRNA core that associates with a nucleic acid programmable DNA binding protein (napDNAbp) having nicking activity, wherein the primer binding site comprises a region of complementarity to a region upstream of a nick site in a second strand of the double-stranded target DNA sequence, and wherein the DNA synthesis template comprises a region of complementarity to a region downstream of the nick site in the second strand of the double-stranded target DNA sequence and comprises one or more nucleotide changes compared to the double-stranded target DNA sequence.
In comparison, instant claim 1 is also drawn to a PEgRNA comprising a spacer sequence capable of targeting the napDNAbp to a target strand of the double stranded DNA sequence, a guide RNA core capable of binding to the napDNAbp, and a DNA extension arm comprising a DNA synthesis template encoding the desired nucleotide change and a primer binding site capable of hybridizing to the single stranded DNA having the 3’ end.
Regarding the additional limitations of the instant claim set, as set forth above, the prior art discloses these limitations.
With regard to claim 1, which recites “a method for editing a double-stranded DNA sequence, the method comprising: contacting the double-stranded DNA sequence with a complex comprising a prime editor and a prime editing guide RNA (PEgRNA), wherein the prime editor comprises (i) a nucleic acid programmable DNA binding protein (napDNAbp) capable of nicking a non-target strand of the double stranded DNA sequence to produce a single strand[ed] DNA having a 3’ end and (ii) a polymerase, and wherein the PEgRNA comprises: (i) a spacer sequence capable of targeting the napDNAbp to a target strand of the double stranded DNA sequence, (ii) a gRNA core capable of binding to the napDNAbp, and (iii) a DNA extension arm comprising a DNA synthesis template encoding the desired nucleotide change and a primer binding site capable of hybridizing to the single strand[ed] DNA having the 3’ end,” Fraser discloses methods for high-efficiency genome editing, wherein the method comprises providing retron-guide RNA cassettes (abstract). The retron-guide RNA cassettes of Fraser read on the instantly claimed PEgRNA, as they comprise a retron transcript (i.e. the extension arm of the claim) and a guide RNA molecule comprising a spacer complementary to a first strand and a guide RNA core capable of binding to the Cas napDNAbp taught therein (paragraphs [0003], [0006], [0009], [0010], and [0127]). As set forth above, patent ‘652 also recites PEgRNAs. Fraser explicitly discloses that the retron transcript of the retron-guide RNA cassettes taught therein is capable of self-priming reverse transcription by a reverse transcriptase (paragraph [0020]). The reverse transcriptase of Fraser (see paragraphs [0106], [0147], [0148], and [0154]) reads on the instantly claimed polymerase (see paragraph [0010] of the instant specification), while the Cas nickase disclosed therein (see paragraphs [0066], [0138], [0142], and [0143]) read on the instantly claimed napDNAbp capable of nicking a strand of the double stranded DNA sequence to produce a single stranded DNA having a 3’ end. Thus, Fraser discloses each and every limitation of the instantly claimed method with the exception of specifically nicking the non-target strand. This deficiency is cured by Komor.
While the disclosure of Fraser is drawn to coordinated actions of a Cas nickase (paragraphs [0066], [0138], [0142], and [0143]) and a reverse transcriptase (paragraphs [0020], [0106], [0147], [0148], and [0154]), Cas nickase activity has been coordinated with other effector proteins. Komor discloses base editors, which comprise a nickase Cas9 such as nSpCas9 and nSaCas9 fused to a base editing enzyme such as APOBEC1, wherein the Cas9 nickase nicks the non-edited strand in order to facilitate base editing of the targeted residue in the edited (i.e. targeted) strand (Figure 2). While the disclosure of Fraser and Komor differ in their effects, both disclosures are drawn to coordinated actions of a Cas nickase and an effector protein for purposes of editing a targeted nucleic acid. Therefore, based on these disclosures, one of ordinary skill in the art would have been motivated, prior to the effective filing date of the instant application, to apply the principles disclosed in Komor to the methods of Fraser such that the Cas nickase disclosed therein specifically nicks the non-targeted strand in order to specifically edit the targeted strand, as set forth in greater detail below.
With regard to claim 11, which recites “the napDNAbp [of the method of claim 1] is a Cas9 nickase (nCas9),” as set forth above, Fraser discloses coordinated actions of a Cas nickase and a reverse transcriptase, wherein the Cas nickase is specifically a Cas9 nickase (paragraph [0143]). This is also recited at patented claims 27 and 29.
With regard to claim 13, which recites “the napDNAbp [of the method of claim 1] comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 137,” as set forth above, Fraser discloses that a Cas9 nickase may be used in the editing methods disclosed therein. However, Fraser is silent as to the sequence of said Cas9 nickase. This deficiency is cured by Huang.
Huang discloses that an exemplary nicking Cas9 protein is Cas9 H840A, which corresponds to SEQ ID NO: 6 taught therein (paragraphs [0077] and [0080]). As shown in the alignment of the Appendix, SEQ ID NO: 6 of Huang comprises 100% identity to instant SEQ ID NO: 137. While the patented application is silent as to the instantly claimed sequence, this sequence reads on the limitations of patented claim 28.
With regard to claim 14, which recites “the polymerase [of the method of claim 1] is a reverse transcriptase,” as set forth above, Fraser discloses coordinated actions of a Cas nickase and a reverse transcriptase (paragraphs [0009], [0106], [0147], [0148], and [0154]).
With regard to claim 15, which recites “the reverse transcriptase [of the method of claim 14] comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 139,” as set forth above, Fraser discloses that a reverse transcriptase may be used in the editing methods disclosed therein. However, Fraser is silent as to the sequence of said reverse transcriptase. This deficiency is cured by Ohtsubo, Yin, and Uniprot, as set forth below.
Ohtsubo teaches that the Moloney murine leukemia virus (MMLV) reverse transcriptase (MMLV-RT) is widely used for cDNA synthesis (i.e. RNA to DNA) but also has DNA polymerase activity when using DNA as a template (page 1, paragraph 3 of the introduction). Per Yin, adding DNA residues to CRISPR guide RNAs significantly reduces off-target genome editing and reduces the associated cost (abstract). Therefore, prior to the effective filing date of the instant invention, one of ordinary skill in the art would have been motivated to generate a guide RNA per the teachings of Yin that comprises DNA residues. Therefore, the reverse transcriptase disclosed in Fraser must be capable of synthesizing DNA from both DNA and RNA in order to utilize both chimeric guide RNAs and standard guide RNAs efficiently. As set forth above, Ohtsubo teaches that MMLV-RT is uniquely suited to both RNA to DNA synthesis and DNA to DNA synthesis. Accordingly, one of ordinary skill in the art would have been motivated to utilize MMLV-RT in the methods of Fraser. UniProt teaches the sequence of MMLV-RT, which comprises 98.8% sequence identity to instant SEQ ID NO: 139, as shown in the alignment of the Appendix.
With regard to claim 16, which recites “the DNA extension arm [of the method of claim 1] is at the 3’ end of the PEgRNA,” Fraser further discloses that the retron-guide RNA cassette taught therein may be structured such that the gRNA coding region is located 3’ or 5’ of the retron (paragraphs [0010] and [0102]).
With regard to claim 82, which recites “the prime editor [of the method of claim 1] is a fusion protein,” as set forth above, Fraser discloses coordinated actions of a Cas nickase and a reverse transcriptase for purposes of editing a nucleic acid sequence. However, Fraser does not explicitly disclose fusion of the Cas nickase and the reverse transcriptase taught therein. This deficiency is cured by Komor. Komor discloses base editors, which comprise a nickase Cas9 such as nSpCas9 and nSaCas9 fused to a base editing enzyme such as APOBEC1 (Figure 2). While the disclosure of Fraser and Komor differ in their effects, both disclosures are drawn to coordinated actions of a Cas nickase and an effector protein for purposes of editing a targeted nucleic acid. Therefore, based on these disclosures, one of ordinary skill in the art would have been motivated, prior to the effective filing date of the instant application, to apply the principles disclosed in Komor to the methods of Fraser such that the Cas nickase disclosed therein is fused to the reverse transcriptase disclosed therein.
With regard to claim 83, which recites “the napDNAbp [of the method of claim 82] is N-terminal of the polymerase in the fusion protein,” as set forth above, Fraser and Komor collectively disclose a fusion protein comprising a Cas nickase and a reverse transcriptase. However, neither specifically teaches the architecture of said fusion protein. Given that the fusion protein collectively disclosed by Fraser and Komor comprises only two protein components, there are only two possible arrangements: one in which the Cas nickase is N-terminal of the reverse transcriptase in the fusion protein, and one in which the Cas nickase is C-terminal of the reverse transcriptase in the fusion protein. Per MPEP § 2144.04(VI)(C), it is not inventive to rearrange parts. Given that there are only a small, finite number of possible arrangements, it is not considered inventive to merely rearrange these components.
With regard to claim 84, which recites “the prime editor [of the method of claim 83] further comprises a peptide linker between the napDNAbp and the polymerase,” as set forth above, Fraser and Komor collectively disclose the method of claim 83. However, neither Fraser nor Komor disclose the peptide linker of instant claim 84. This deficiency is cured by Zhang.
Zhang discloses methods and compositions for targeting and editing nucleic acids, particularly RNA targeted and edited via a Cas13 fusion protein comprising a Cas13 fused to an editing enzyme such as ADAR (abstract; paragraph [0047]; Figure 2). While the methods of Zhang are drawn to RNA editing and not DNA editing, Zhang nonetheless discloses coordinated activity of a Cas enzyme and an editing enzyme, as in the instant application, Fraser, and Komor. Zhang further discloses that the fusion proteins taught therein may be linked via a peptide linker (paragraphs [0200] and [0201]). Thus, Zhang establishes that Cas proteins may be fused to effector proteins via a peptide linker, as instantly claimed.
With regard to claim 85, which recites “the polymerase [of the method of claim 1] is not connected to the napDNAbp in a fusion protein,” as set forth above, Fraser discloses coordinated actions of a Cas9 nickase and a reverse transcriptase for purposes of editing a nucleic acid sequence. Fraser further discloses that the reverse transcriptase and Cas effector disclosed therein may be provided separately (paragraphs [0026] and [0027]).
With regard to claim 86, which recites “the PEgRNA further comprises an RNA-binding protein recruitment domain,” as set forth above, the retron-guide cassette of Fraser reads on the instantly claimed PEgRNA. Zhang further discloses that the guide molecule taught therein comprises an aptamer sequence capable of binding to an RNA-binding adaptor protein such as MS2 (paragraphs [0011], [0315], and [0316), which reads on the instantly claimed RNA-binding protein recruitment domain.
With regard to claim 87, which recites “the RNA-binding protein recruitment domain [of the method of claim 86] is an MS2 aptamer,” as set forth above, Zhang further discloses that the guide molecule taught therein comprises an aptamer sequence capable of binding to an RNA-binding adaptor protein such as MS2 (paragraphs [0011], [0315], and [0316). This is also recited at patented claims 26 and 30.
With regard to claim 88, which recites “the polymerase [of the method of claim 85] is fused to an RNA-binding protein domain,” Zhang further discloses that the adenosine deaminase effector taught therein may be fused to RNA-binding protein MS2 coat protein (paragraphs [0315] and [0316]). While the adenosine deaminase effector of Zhang does not read on the instantly claimed polymerase, the disclosure of Zhang is applied to establish that principles of coordinating activity of a Cas protein and an editing enzyme, as in the instant application, Fraser, and Komor. Therefore, Zhang teaches that the effector/editing enzyme taught therein may be fused to an RNA-binding protein such as MS2 coat protein for purposes of implementing orthogonal gene editing (paragraphs [0315], [0316], and [0325]).
With regard to claim 89, which recites “the polymerase [of the method of claim 86] is fused to an MS2cp domain,” as set forth above, Zhang further discloses that the adenosine deaminase effector taught therein may be fused to RNA-binding protein MS2 coat protein for purposes of implementing orthogonal gene editing (paragraphs [0315], [0316], and [0325]).
With regard to claim 90, which recites “the double stranded DNA [of the method of claim 1] is in a cell,” Fraser discloses that the methods taught therein may be practiced within a genome of a host cell, wherein the host cell is transformed with a vector carrying the machinery set forth above and is subsequently edited (paragraph [0024]).
With regard to claim 92, which recites “the method of claim 90, further comprising delivering an mRNA encoding the prime editor into the cell,” as set forth above, Fraser and Komor collectively disclose the instantly claimed prime editor. While Fraser discloses that the reverse transcriptase and Cas effector taught therein may be expressed from a vector or plasmid (paragraphs [0026], [0027], and [0145]), Fraser does not explicitly disclose that these components are delivered as an mRNA. This deficiency is cured by Zhang, which discloses that the editing compositions disclosed therein (including Cas-enzyme fusions) may be delivered as mRNA molecules encoding all of the components thereof (paragraphs [0318] and [0319]).
With regard to claim 93, which recites “the mRNA and the pegRNA [of the method of claim 92] are delivered via a lipid nanoparticle,” Zhang further discloses that the mRNA encoding the components of the system taught therein (and set forth above) may be delivered via one or more lipid nanoparticles (paragraphs [0318] and [0319]).
With regard to claim 94, which recites “the method of claim 1, further comprising contacting the double stranded DNA with a complex comprising the prime editor and a nicking guide RNA (ngRNA),” Fraser further discloses that providing at least two DNA-targeting RNAs targeting opposite DNA strands facilitates a double-nicked double-strand break that favors HDR and decreases the frequency of insertion/deletion mutations at off-target DNA sites (paragraph [0143]). Per Fraser, two nickases of the invention disclosed therein may be utilized to create two single-stranded breaks on opposite strands of a target DNA, as set forth above (paragraph [0066]).
With regard to claim 95, which recites “the ngRNA [of the method of claim 94] comprises a[n] ngRNA spacer sequence capable of targeting the napDNAbp to the non-target strand of the double-stranded DNA sequence,” as set forth above, Fraser further discloses that providing at least two DNA-targeting RNAs targeting opposite DNA strands facilitates a double-nicked double-strand break that favors HDR and decreases the frequency of insertion/deletion mutations at off-target DNA sites (paragraph [0143]). Per Fraser, two nickases of the invention disclosed therein may be utilized to create two single-stranded breaks on opposite strands of a target DNA (paragraph [0066]). The DNA-targeting RNAs of Fraser read on the guide RNAs of Fraser, which are disclosed to comprise a crRNA spacer sequence that is complementary to a target nucleic acid sequence (paragraph [0127]).
Given that patent ‘303 also discloses PEgRNAs, as in the instant application, that Fraser and Komor collectively disclose a method of editing a target genome utilizing said PEgRNAs, that Huang and UniProt disclose the napDNAbp and MMLV-RT sequences of the instant and patented applications, and that Zhang discloses the utility of including an MS2 coat protein and associated binding aptamer thereof in such systems, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to utilize the PEgRNA of patent ‘303 in the systems collectively disclosed by the prior art, as well as to include an MS2 coat protein and associated binding aptamer thereof to predictably prime edit a double-stranded DNA, as instantly claimed. One would have been motivated to make such a modification in order to receive the expected benefit of practicing prime editing.
Claims 1, 11, 13-16, 82-90, and 92-95 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 20-27, 30, and 31 of U.S. Patent No. 12,509,680 B2 (as cited in the IDS filed 07/15/2026) in view of WO 2018/049168 A1 (hereinafter Fraser; as cited in the IDS filed 08/24/2023), Komor et al., 2017 (hereinafter Komor; as cited in the IDS filed 08/24/2023), US 2016/0289673 A1 (hereinafter Huang), Ohtsubo et al., 2017 (hereinafter Ohtsubo), Yin et al., 2018 (hereinafter Yin), UniProt Entry P03355 (hereinafter UniProt; first entered 07/21/1986), and WO 2019/005884 (hereinafter Zhang; as cited in the IDS filed 08/24/2023).
MPEP 804 II B 1 states:
The specification can be used as a dictionary to learn the meaning of a term in the patent claim. Toro Co. v. White Consol. Indus., Inc., 199 F.3d 1295, 1299, 53 USPQ2d 1065, 1067 (Fed. Cir. 1999)… Further, those portions of the specification which provide support for the patent claims may also be examined and considered when addressing the issue of whether a claim in the application defines an obvious variation of an invention claimed in the patent. In re Vogel, 422 F.2d 438, 441-42, 164 USPQ 619, 622 (CCPA 1970).
The following rejections are in view of the decision of the Court of Appeals for the Federal Circuit in Pfizer Inc, v Teva pharmaceuticals USA Inc., 86 USPQ2d 1001, at page 1008 (March 2008), which indicates that there is no patentable distinction between claims to a product and a method of using that product disclosed in the specification of the application and that the preclusion of such a double patenting rejection under 35 USC 121 does not apply where the present application is other than a divisional application of the patent application containing such patentably indistinct claims.
Claim 1 of patent ‘303 is drawn to a multiplex prime editing system, said system comprising a prime editor comprising a napDNAbp that is a nickase configured to cut a non-target strand of a double-stranded DNA sequence and a reverse transcriptase domain, as well as a prime editing guide RNA comprising a spacer sequence, an RNA extension arm that comprises a DNA synthesis template and a primer binding site, and a gRNA core that associates with a nucleic acid programmable DNA binding protein (napDNAbp) having nicking activity, wherein the primer binding site comprises a region of complementarity to a region upstream of a nick site in a second strand of the double-stranded target DNA sequence, and wherein the DNA synthesis template comprises a region of complementarity to a region downstream of the nick site in the second strand of the double-stranded target DNA sequence and comprises one or more nucleotide changes compared to the double-stranded target DNA sequence.
In comparison, instant claim 1 is also drawn to a PEgRNA comprising a spacer sequence capable of targeting the napDNAbp to a target strand of the double stranded DNA sequence, a guide RNA core capable of binding to the napDNAbp, and a DNA extension arm comprising a DNA synthesis template encoding the desired nucleotide change and a primer binding site capable of hybridizing to the single stranded DNA having the 3’ end.
Regarding the additional limitations of the instant claim set, as set forth above, the prior art discloses these limitations.
With regard to claim 1, which recites “a method for editing a double-stranded DNA sequence, the method comprising: contacting the double-stranded DNA sequence with a complex comprising a prime editor and a prime editing guide RNA (PEgRNA), wherein the prime editor comprises (i) a nucleic acid programmable DNA binding protein (napDNAbp) capable of nicking a non-target strand of the double stranded DNA sequence to produce a single strand[ed] DNA having a 3’ end and (ii) a polymerase, and wherein the PEgRNA comprises: (i) a spacer sequence capable of targeting the napDNAbp to a target strand of the double stranded DNA sequence, (ii) a gRNA core capable of binding to the napDNAbp, and (iii) a DNA extension arm comprising a DNA synthesis template encoding the desired nucleotide change and a primer binding site capable of hybridizing to the single strand[ed] DNA having the 3’ end,” Fraser discloses methods for high-efficiency genome editing, wherein the method comprises providing retron-guide RNA cassettes (abstract). The retron-guide RNA cassettes of Fraser read on the instantly claimed PEgRNA, as they comprise a retron transcript (i.e. the extension arm of the claim) and a guide RNA molecule comprising a spacer complementary to a first strand and a guide RNA core capable of binding to the Cas napDNAbp taught therein (paragraphs [0003], [0006], [0009], [0010], and [0127]). As set forth above, patent ‘652 also recites PEgRNAs. Fraser explicitly discloses that the retron transcript of the retron-guide RNA cassettes taught therein is capable of self-priming reverse transcription by a reverse transcriptase (paragraph [0020]). The reverse transcriptase of Fraser (see paragraphs [0106], [0147], [0148], and [0154]) reads on the instantly claimed polymerase (see paragraph [0010] of the instant specification), while the Cas nickase disclosed therein (see paragraphs [0066], [0138], [0142], and [0143]) read on the instantly claimed napDNAbp capable of nicking a strand of the double stranded DNA sequence to produce a single stranded DNA having a 3’ end. Thus, Fraser discloses each and every limitation of the instantly claimed method with the exception of specifically nicking the non-target strand. This deficiency is cured by Komor.
While the disclosure of Fraser is drawn to coordinated actions of a Cas nickase (paragraphs [0066], [0138], [0142], and [0143]) and a reverse transcriptase (paragraphs [0020], [0106], [0147], [0148], and [0154]), Cas nickase activity has been coordinated with other effector proteins. Komor discloses base editors, which comprise a nickase Cas9 such as nSpCas9 and nSaCas9 fused to a base editing enzyme such as APOBEC1, wherein the Cas9 nickase nicks the non-edited strand in order to facilitate base editing of the targeted residue in the edited (i.e. targeted) strand (Figure 2). While the disclosure of Fraser and Komor differ in their effects, both disclosures are drawn to coordinated actions of a Cas nickase and an effector protein for purposes of editing a targeted nucleic acid. Therefore, based on these disclosures, one of ordinary skill in the art would have been motivated, prior to the effective filing date of the instant application, to apply the principles disclosed in Komor to the methods of Fraser such that the Cas nickase disclosed therein specifically nicks the non-targeted strand in order to specifically edit the targeted strand, as set forth in greater detail below.
With regard to claim 11, which recites “the napDNAbp [of the method of claim 1] is a Cas9 nickase (nCas9),” as set forth above, Fraser discloses coordinated actions of a Cas nickase and a reverse transcriptase, wherein the Cas nickase is specifically a Cas9 nickase (paragraph [0143]). This is also recited at patented claims 21-24
With regard to claim 13, which recites “the napDNAbp [of the method of claim 1] comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 137,” as set forth above, Fraser discloses that a Cas9 nickase may be used in the editing methods disclosed therein. However, Fraser is silent as to the sequence of said Cas9 nickase. This deficiency is cured by Huang.
Huang discloses that an exemplary nicking Cas9 protein is Cas9 H840A, which corresponds to SEQ ID NO: 6 taught therein (paragraphs [0077] and [0080]). As shown in the alignment of the Appendix, SEQ ID NO: 6 of Huang comprises 100% identity to instant SEQ ID NO: 137. While the patented application is silent as to the instantly claimed sequence, this sequence reads on the limitations of patented claim 31.
With regard to claim 14, which recites “the polymerase [of the method of claim 1] is a reverse transcriptase,” as set forth above, Fraser discloses coordinated actions of a Cas nickase and a reverse transcriptase (paragraphs [0009], [0106], [0147], [0148], and [0154]).
With regard to claim 15, which recites “the reverse transcriptase [of the method of claim 14] comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 139,” as set forth above, Fraser discloses that a reverse transcriptase may be used in the editing methods disclosed therein. However, Fraser is silent as to the sequence of said reverse transcriptase. This deficiency is cured by Ohtsubo, Yin, and Uniprot, as set forth below.
Ohtsubo teaches that the Moloney murine leukemia virus (MMLV) reverse transcriptase (MMLV-RT) is widely used for cDNA synthesis (i.e. RNA to DNA) but also has DNA polymerase activity when using DNA as a template (page 1, paragraph 3 of the introduction). Per Yin, adding DNA residues to CRISPR guide RNAs significantly reduces off-target genome editing and reduces the associated cost (abstract). Therefore, prior to the effective filing date of the instant invention, one of ordinary skill in the art would have been motivated to generate a guide RNA per the teachings of Yin that comprises DNA residues. Therefore, the reverse transcriptase disclosed in Fraser must be capable of synthesizing DNA from both DNA and RNA in order to utilize both chimeric guide RNAs and standard guide RNAs efficiently. As set forth above, Ohtsubo teaches that MMLV-RT is uniquely suited to both RNA to DNA synthesis and DNA to DNA synthesis. Accordingly, one of ordinary skill in the art would have been motivated to utilize MMLV-RT in the methods of Fraser. UniProt teaches the sequence of MMLV-RT, which comprises 98.8% sequence identity to instant SEQ ID NO: 139, as shown in the alignment of the Appendix. While patented claims 25 and 25 also recite M-MLV RT, they are silent as to the instantly claimed sequence.
With regard to claim 16, which recites “the DNA extension arm [of the method of claim 1] is at the 3’ end of the PEgRNA,” Fraser further discloses that the retron-guide RNA cassette taught therein may be structured such that the gRNA coding region is located 3’ or 5’ of the retron (paragraphs [0010] and [0102]).
With regard to claim 82, which recites “the prime editor [of the method of claim 1] is a fusion protein,” as set forth above, Fraser discloses coordinated actions of a Cas nickase and a reverse transcriptase for purposes of editing a nucleic acid sequence. However, Fraser does not explicitly disclose fusion of the Cas nickase and the reverse transcriptase taught therein. This deficiency is cured by Komor. Komor discloses base editors, which comprise a nickase Cas9 such as nSpCas9 and nSaCas9 fused to a base editing enzyme such as APOBEC1 (Figure 2). While the disclosure of Fraser and Komor differ in their effects, both disclosures are drawn to coordinated actions of a Cas nickase and an effector protein for purposes of editing a targeted nucleic acid. Therefore, based on these disclosures, one of ordinary skill in the art would have been motivated, prior to the effective filing date of the instant application, to apply the principles disclosed in Komor to the methods of Fraser such that the Cas nickase disclosed therein is fused to the reverse transcriptase disclosed therein. This is also recited at patented claim 27.
With regard to claim 83, which recites “the napDNAbp [of the method of claim 82] is N-terminal of the polymerase in the fusion protein,” as set forth above, Fraser and Komor collectively disclose a fusion protein comprising a Cas nickase and a reverse transcriptase. However, neither specifically teaches the architecture of said fusion protein. Given that the fusion protein collectively disclosed by Fraser and Komor comprises only two protein components, there are only two possible arrangements: one in which the Cas nickase is N-terminal of the reverse transcriptase in the fusion protein, and one in which the Cas nickase is C-terminal of the reverse transcriptase in the fusion protein. Per MPEP § 2144.04(VI)(C), it is not inventive to rearrange parts. Given that there are only a small, finite number of possible arrangements, it is not considered inventive to merely rearrange these components.
With regard to claim 84, which recites “the prime editor [of the method of claim 83] further comprises a peptide linker between the napDNAbp and the polymerase,” as set forth above, Fraser and Komor collectively disclose the method of claim 83. However, neither Fraser nor Komor disclose the peptide linker of instant claim 84. This deficiency is cured by Zhang.
Zhang discloses methods and compositions for targeting and editing nucleic acids, particularly RNA targeted and edited via a Cas13 fusion protein comprising a Cas13 fused to an editing enzyme such as ADAR (abstract; paragraph [0047]; Figure 2). While the methods of Zhang are drawn to RNA editing and not DNA editing, Zhang nonetheless discloses coordinated activity of a Cas enzyme and an editing enzyme, as in the instant application, Fraser, and Komor. Zhang further discloses that the fusion proteins taught therein may be linked via a peptide linker (paragraphs [0200] and [0201]). Thus, Zhang establishes that Cas proteins may be fused to effector proteins via a peptide linker, as instantly claimed.
With regard to claim 85, which recites “the polymerase [of the method of claim 1] is not connected to the napDNAbp in a fusion protein,” as set forth above, Fraser discloses coordinated actions of a Cas9 nickase and a reverse transcriptase for purposes of editing a nucleic acid sequence. Fraser further discloses that the reverse transcriptase and Cas effector disclosed therein may be provided separately (paragraphs [0026] and [0027]).
With regard to claim 86, which recites “the PEgRNA further comprises an RNA-binding protein recruitment domain,” as set forth above, the retron-guide cassette of Fraser reads on the instantly claimed PEgRNA. Zhang further discloses that the guide molecule taught therein comprises an aptamer sequence capable of binding to an RNA-binding adaptor protein such as MS2 (paragraphs [0011], [0315], and [0316), which reads on the instantly claimed RNA-binding protein recruitment domain.
With regard to claim 87, which recites “the RNA-binding protein recruitment domain [of the method of claim 86] is an MS2 aptamer,” as set forth above, Zhang further discloses that the guide molecule taught therein comprises an aptamer sequence capable of binding to an RNA-binding adaptor protein such as MS2 (paragraphs [0011], [0315], and [0316). This is also recited at patented claims 20 and 30.
With regard to claim 88, which recites “the polymerase [of the method of claim 85] is fused to an RNA-binding protein domain,” Zhang further discloses that the adenosine deaminase effector taught therein may be fused to RNA-binding protein MS2 coat protein (paragraphs [0315] and [0316]). While the adenosine deaminase effector of Zhang does not read on the instantly claimed polymerase, the disclosure of Zhang is applied to establish that principles of coordinating activity of a Cas protein and an editing enzyme, as in the instant application, Fraser, and Komor. Therefore, Zhang teaches that the effector/editing enzyme taught therein may be fused to an RNA-binding protein such as MS2 coat protein for purposes of implementing orthogonal gene editing (paragraphs [0315], [0316], and [0325]).
With regard to claim 89, which recites “the polymerase [of the method of claim 86] is fused to an MS2cp domain,” as set forth above, Zhang further discloses that the adenosine deaminase effector taught therein may be fused to RNA-binding protein MS2 coat protein for purposes of implementing orthogonal gene editing (paragraphs [0315], [0316], and [0325]).
With regard to claim 90, which recites “the double stranded DNA [of the method of claim 1] is in a cell,” Fraser discloses that the methods taught therein may be practiced within a genome of a host cell, wherein the host cell is transformed with a vector carrying the machinery set forth above and is subsequently edited (paragraph [0024]).
With regard to claim 92, which recites “the method of claim 90, further comprising delivering an mRNA encoding the prime editor into the cell,” as set forth above, Fraser and Komor collectively disclose the instantly claimed prime editor. While Fraser discloses that the reverse transcriptase and Cas effector taught therein may be expressed from a vector or plasmid (paragraphs [0026], [0027], and [0145]), Fraser does not explicitly disclose that these components are delivered as an mRNA. This deficiency is cured by Zhang, which discloses that the editing compositions disclosed therein (including Cas-enzyme fusions) may be delivered as mRNA molecules encoding all of the components thereof (paragraphs [0318] and [0319]).
With regard to claim 93, which recites “the mRNA and the pegRNA [of the method of claim 92] are delivered via a lipid nanoparticle,” Zhang further discloses that the mRNA encoding the components of the system taught therein (and set forth above) may be delivered via one or more lipid nanoparticles (paragraphs [0318] and [0319]).
With regard to claim 94, which recites “the method of claim 1, further comprising contacting the double stranded DNA with a complex comprising the prime editor and a nicking guide RNA (ngRNA),” Fraser further discloses that providing at least two DNA-targeting RNAs targeting opposite DNA strands facilitates a double-nicked double-strand break that favors HDR and decreases the frequency of insertion/deletion mutations at off-target DNA sites (paragraph [0143]). Per Fraser, two nickases of the invention disclosed therein may be utilized to create two single-stranded breaks on opposite strands of a target DNA, as set forth above (paragraph [0066]).
With regard to claim 95, which recites “the ngRNA [of the method of claim 94] comprises a[n] ngRNA spacer sequence capable of targeting the napDNAbp to the non-target strand of the double-stranded DNA sequence,” as set forth above, Fraser further discloses that providing at least two DNA-targeting RNAs targeting opposite DNA strands facilitates a double-nicked double-strand break that favors HDR and decreases the frequency of insertion/deletion mutations at off-target DNA sites (paragraph [0143]). Per Fraser, two nickases of the invention disclosed therein may be utilized to create two single-stranded breaks on opposite strands of a target DNA (paragraph [0066]). The DNA-targeting RNAs of Fraser read on the guide RNAs of Fraser, which are disclosed to comprise a crRNA spacer sequence that is complementary to a target nucleic acid sequence (paragraph [0127]).
Given that patent ‘680 also discloses PEgRNAs, as in the instant application, that Fraser and Komor collectively disclose a method of editing a target genome utilizing said PEgRNAs, that Huang and UniProt disclose the napDNAbp and MMLV-RT sequences of the instant and patented applications, and that Zhang discloses the utility of including an MS2 coat protein and associated binding aptamer thereof in such systems, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to utilize the PEgRNA of patent ‘680 in the systems collectively disclosed by the prior art, as well as to include an MS2 coat protein and associated binding aptamer thereof to predictably prime edit a double-stranded DNA, as instantly claimed. One would have been motivated to make such a modification in order to receive the expected benefit of practicing prime editing.
Claims 1, 11, 13-16, 82-90, and 92-95 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 2, and 55-57of Application No. 18/064,738 (corresponds to US 2024/0229077 A1; as cited in the IDS filed 07/15/2026) in view of WO 2018/049168 A1 (hereinafter Fraser; as cited in the IDS filed 08/24/2023), Komor et al., 2017 (hereinafter Komor; as cited in the IDS filed 08/24/2023), US 2016/0289673 A1 (hereinafter Huang), Ohtsubo et al., 2017 (hereinafter Ohtsubo), Yin et al., 2018 (hereinafter Yin), UniProt Entry P03355 (hereinafter UniProt; first entered 07/21/1986), and WO 2019/005884 (hereinafter Zhang; as cited in the IDS filed 08/24/2023).
MPEP 804 II B 1 states:
The specification can be used as a dictionary to learn the meaning of a term in the patent claim. Toro Co. v. White Consol. Indus., Inc., 199 F.3d 1295, 1299, 53 USPQ2d 1065, 1067 (Fed. Cir. 1999)… Further, those portions of the specification which provide support for the patent claims may also be examined and considered when addressing the issue of whether a claim in the application defines an obvious variation of an invention claimed in the patent. In re Vogel, 422 F.2d 438, 441-42, 164 USPQ 619, 622 (CCPA 1970).
The following rejections are in view of the decision of the Court of Appeals for the Federal Circuit in Pfizer Inc, v Teva pharmaceuticals USA Inc., 86 USPQ2d 1001, at page 1008 (March 2008), which indicates that there is no patentable distinction between claims to a product and a method of using that product disclosed in the specification of the application and that the preclusion of such a double patenting rejection under 35 USC 121 does not apply where the present application is other than a divisional application of the patent application containing such patentably indistinct claims.
Copending claim 1 is drawn to a PEgRNA that reads on the PEgRNA of the instant application. Regarding the additional limitations of the instant claim set, as set forth above, the prior art discloses these limitations.
With regard to claim 1, which recites “a method for editing a double-stranded DNA sequence, the method comprising: contacting the double-stranded DNA sequence with a complex comprising a prime editor and a prime editing guide RNA (PEgRNA), wherein the prime editor comprises (i) a nucleic acid programmable DNA binding protein (napDNAbp) capable of nicking a non-target strand of the double stranded DNA sequence to produce a single strand[ed] DNA having a 3’ end and (ii) a polymerase, and wherein the PEgRNA comprises: (i) a spacer sequence capable of targeting the napDNAbp to a target strand of the double stranded DNA sequence, (ii) a gRNA core capable of binding to the napDNAbp, and (iii) a DNA extension arm comprising a DNA synthesis template encoding the desired nucleotide change and a primer binding site capable of hybridizing to the single strand[ed] DNA having the 3’ end,” Fraser discloses methods for high-efficiency genome editing, wherein the method comprises providing retron-guide RNA cassettes (abstract). The retron-guide RNA cassettes of Fraser read on the instantly claimed PEgRNA, as they comprise a retron transcript (i.e. the extension arm of the claim) and a guide RNA molecule comprising a spacer complementary to a first strand and a guide RNA core capable of binding to the Cas napDNAbp taught therein (paragraphs [0003], [0006], [0009], [0010], and [0127]). As set forth above, patent ‘652 also recites PEgRNAs. Fraser explicitly discloses that the retron transcript of the retron-guide RNA cassettes taught therein is capable of self-priming reverse transcription by a reverse transcriptase (paragraph [0020]). The reverse transcriptase of Fraser (see paragraphs [0106], [0147], [0148], and [0154]) reads on the instantly claimed polymerase (see paragraph [0010] of the instant specification), while the Cas nickase disclosed therein (see paragraphs [0066], [0138], [0142], and [0143]) read on the instantly claimed napDNAbp capable of nicking a strand of the double stranded DNA sequence to produce a single stranded DNA having a 3’ end. Thus, Fraser discloses each and every limitation of the instantly claimed method with the exception of specifically nicking the non-target strand. This deficiency is cured by Komor.
While the disclosure of Fraser is drawn to coordinated actions of a Cas nickase (paragraphs [0066], [0138], [0142], and [0143]) and a reverse transcriptase (paragraphs [0020], [0106], [0147], [0148], and [0154]), Cas nickase activity has been coordinated with other effector proteins. Komor discloses base editors, which comprise a nickase Cas9 such as nSpCas9 and nSaCas9 fused to a base editing enzyme such as APOBEC1, wherein the Cas9 nickase nicks the non-edited strand in order to facilitate base editing of the targeted residue in the edited (i.e. targeted) strand (Figure 2). While the disclosure of Fraser and Komor differ in their effects, both disclosures are drawn to coordinated actions of a Cas nickase and an effector protein for purposes of editing a targeted nucleic acid. Therefore, based on these disclosures, one of ordinary skill in the art would have been motivated, prior to the effective filing date of the instant application, to apply the principles disclosed in Komor to the methods of Fraser such that the Cas nickase disclosed therein specifically nicks the non-targeted strand in order to specifically edit the targeted strand, as set forth in greater detail below.
With regard to claim 11, which recites “the napDNAbp [of the method of claim 1] is a Cas9 nickase (nCas9),” as set forth above, Fraser discloses coordinated actions of a Cas nickase and a reverse transcriptase, wherein the Cas nickase is specifically a Cas9 nickase (paragraph [0143]). This is also recited at patented claims 21-24
With regard to claim 13, which recites “the napDNAbp [of the method of claim 1] comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 137,” as set forth above, Fraser discloses that a Cas9 nickase may be used in the editing methods disclosed therein. However, Fraser is silent as to the sequence of said Cas9 nickase. This deficiency is cured by Huang.
Huang discloses that an exemplary nicking Cas9 protein is Cas9 H840A, which corresponds to SEQ ID NO: 6 taught therein (paragraphs [0077] and [0080]). As shown in the alignment of the Appendix, SEQ ID NO: 6 of Huang comprises 100% identity to instant SEQ ID NO: 137.
With regard to claim 14, which recites “the polymerase [of the method of claim 1] is a reverse transcriptase,” as set forth above, Fraser discloses coordinated actions of a Cas nickase and a reverse transcriptase (paragraphs [0009], [0106], [0147], [0148], and [0154]).
With regard to claim 15, which recites “the reverse transcriptase [of the method of claim 14] comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 139,” as set forth above, Fraser discloses that a reverse transcriptase may be used in the editing methods disclosed therein. However, Fraser is silent as to the sequence of said reverse transcriptase. This deficiency is cured by Ohtsubo, Yin, and Uniprot, as set forth below.
Ohtsubo teaches that the Moloney murine leukemia virus (MMLV) reverse transcriptase (MMLV-RT) is widely used for cDNA synthesis (i.e. RNA to DNA) but also has DNA polymerase activity when using DNA as a template (page 1, paragraph 3 of the introduction). Per Yin, adding DNA residues to CRISPR guide RNAs significantly reduces off-target genome editing and reduces the associated cost (abstract). Therefore, prior to the effective filing date of the instant invention, one of ordinary skill in the art would have been motivated to generate a guide RNA per the teachings of Yin that comprises DNA residues. Therefore, the reverse transcriptase disclosed in Fraser must be capable of synthesizing DNA from both DNA and RNA in order to utilize both chimeric guide RNAs and standard guide RNAs efficiently. As set forth above, Ohtsubo teaches that MMLV-RT is uniquely suited to both RNA to DNA synthesis and DNA to DNA synthesis. Accordingly, one of ordinary skill in the art would have been motivated to utilize MMLV-RT in the methods of Fraser. UniProt teaches the sequence of MMLV-RT, which comprises 98.8% sequence identity to instant SEQ ID NO: 139, as shown in the alignment of the Appendix.
With regard to claim 16, which recites “the DNA extension arm [of the method of claim 1] is at the 3’ end of the PEgRNA,” Fraser further discloses that the retron-guide RNA cassette taught therein may be structured such that the gRNA coding region is located 3’ or 5’ of the retron (paragraphs [0010] and [0102]). This is also recited at copending claim 2.
With regard to claim 82, which recites “the prime editor [of the method of claim 1] is a fusion protein,” as set forth above, Fraser discloses coordinated actions of a Cas nickase and a reverse transcriptase for purposes of editing a nucleic acid sequence. However, Fraser does not explicitly disclose fusion of the Cas nickase and the reverse transcriptase taught therein. This deficiency is cured by Komor. Komor discloses base editors, which comprise a nickase Cas9 such as nSpCas9 and nSaCas9 fused to a base editing enzyme such as APOBEC1 (Figure 2). While the disclosure of Fraser and Komor differ in their effects, both disclosures are drawn to coordinated actions of a Cas nickase and an effector protein for purposes of editing a targeted nucleic acid. Therefore, based on these disclosures, one of ordinary skill in the art would have been motivated, prior to the effective filing date of the instant application, to apply the principles disclosed in Komor to the methods of Fraser such that the Cas nickase disclosed therein is fused to the reverse transcriptase disclosed therein.
With regard to claim 83, which recites “the napDNAbp [of the method of claim 82] is N-terminal of the polymerase in the fusion protein,” as set forth above, Fraser and Komor collectively disclose a fusion protein comprising a Cas nickase and a reverse transcriptase. However, neither specifically teaches the architecture of said fusion protein. Given that the fusion protein collectively disclosed by Fraser and Komor comprises only two protein components, there are only two possible arrangements: one in which the Cas nickase is N-terminal of the reverse transcriptase in the fusion protein, and one in which the Cas nickase is C-terminal of the reverse transcriptase in the fusion protein. Per MPEP § 2144.04(VI)(C), it is not inventive to rearrange parts. Given that there are only a small, finite number of possible arrangements, it is not considered inventive to merely rearrange these components.
With regard to claim 84, which recites “the prime editor [of the method of claim 83] further comprises a peptide linker between the napDNAbp and the polymerase,” as set forth above, Fraser and Komor collectively disclose the method of claim 83. However, neither Fraser nor Komor disclose the peptide linker of instant claim 84. This deficiency is cured by Zhang.
Zhang discloses methods and compositions for targeting and editing nucleic acids, particularly RNA targeted and edited via a Cas13 fusion protein comprising a Cas13 fused to an editing enzyme such as ADAR (abstract; paragraph [0047]; Figure 2). While the methods of Zhang are drawn to RNA editing and not DNA editing, Zhang nonetheless discloses coordinated activity of a Cas enzyme and an editing enzyme, as in the instant application, Fraser, and Komor. Zhang further discloses that the fusion proteins taught therein may be linked via a peptide linker (paragraphs [0200] and [0201]). Thus, Zhang establishes that Cas proteins may be fused to effector proteins via a peptide linker, as instantly claimed.
With regard to claim 85, which recites “the polymerase [of the method of claim 1] is not connected to the napDNAbp in a fusion protein,” as set forth above, Fraser discloses coordinated actions of a Cas9 nickase and a reverse transcriptase for purposes of editing a nucleic acid sequence. Fraser further discloses that the reverse transcriptase and Cas effector disclosed therein may be provided separately (paragraphs [0026] and [0027]).
With regard to claim 86, which recites “the PEgRNA further comprises an RNA-binding protein recruitment domain,” as set forth above, the retron-guide cassette of Fraser reads on the instantly claimed PEgRNA. Zhang further discloses that the guide molecule taught therein comprises an aptamer sequence capable of binding to an RNA-binding adaptor protein such as MS2 (paragraphs [0011], [0315], and [0316), which reads on the instantly claimed RNA-binding protein recruitment domain.
With regard to claim 87, which recites “the RNA-binding protein recruitment domain [of the method of claim 86] is an MS2 aptamer,” as set forth above, Zhang further discloses that the guide molecule taught therein comprises an aptamer sequence capable of binding to an RNA-binding adaptor protein such as MS2 (paragraphs [0011], [0315], and [0316). This is also recited at copending claims 55-57.
With regard to claim 88, which recites “the polymerase [of the method of claim 85] is fused to an RNA-binding protein domain,” Zhang further discloses that the adenosine deaminase effector taught therein may be fused to RNA-binding protein MS2 coat protein (paragraphs [0315] and [0316]). While the adenosine deaminase effector of Zhang does not read on the instantly claimed polymerase, the disclosure of Zhang is applied to establish that principles of coordinating activity of a Cas protein and an editing enzyme, as in the instant application, Fraser, and Komor. Therefore, Zhang teaches that the effector/editing enzyme taught therein may be fused to an RNA-binding protein such as MS2 coat protein for purposes of implementing orthogonal gene editing (paragraphs [0315], [0316], and [0325]).
With regard to claim 89, which recites “the polymerase [of the method of claim 86] is fused to an MS2cp domain,” as set forth above, Zhang further discloses that the adenosine deaminase effector taught therein may be fused to RNA-binding protein MS2 coat protein for purposes of implementing orthogonal gene editing (paragraphs [0315], [0316], and [0325]).
With regard to claim 90, which recites “the double stranded DNA [of the method of claim 1] is in a cell,” Fraser discloses that the methods taught therein may be practiced within a genome of a host cell, wherein the host cell is transformed with a vector carrying the machinery set forth above and is subsequently edited (paragraph [0024]).
With regard to claim 92, which recites “the method of claim 90, further comprising delivering an mRNA encoding the prime editor into the cell,” as set forth above, Fraser and Komor collectively disclose the instantly claimed prime editor. While Fraser discloses that the reverse transcriptase and Cas effector taught therein may be expressed from a vector or plasmid (paragraphs [0026], [0027], and [0145]), Fraser does not explicitly disclose that these components are delivered as an mRNA. This deficiency is cured by Zhang, which discloses that the editing compositions disclosed therein (including Cas-enzyme fusions) may be delivered as mRNA molecules encoding all of the components thereof (paragraphs [0318] and [0319]).
With regard to claim 93, which recites “the mRNA and the pegRNA [of the method of claim 92] are delivered via a lipid nanoparticle,” Zhang further discloses that the mRNA encoding the components of the system taught therein (and set forth above) may be delivered via one or more lipid nanoparticles (paragraphs [0318] and [0319]).
With regard to claim 94, which recites “the method of claim 1, further comprising contacting the double stranded DNA with a complex comprising the prime editor and a nicking guide RNA (ngRNA),” Fraser further discloses that providing at least two DNA-targeting RNAs targeting opposite DNA strands facilitates a double-nicked double-strand break that favors HDR and decreases the frequency of insertion/deletion mutations at off-target DNA sites (paragraph [0143]). Per Fraser, two nickases of the invention disclosed therein may be utilized to create two single-stranded breaks on opposite strands of a target DNA, as set forth above (paragraph [0066]).
With regard to claim 95, which recites “the ngRNA [of the method of claim 94] comprises a[n] ngRNA spacer sequence capable of targeting the napDNAbp to the non-target strand of the double-stranded DNA sequence,” as set forth above, Fraser further discloses that providing at least two DNA-targeting RNAs targeting opposite DNA strands facilitates a double-nicked double-strand break that favors HDR and decreases the frequency of insertion/deletion mutations at off-target DNA sites (paragraph [0143]). Per Fraser, two nickases of the invention disclosed therein may be utilized to create two single-stranded breaks on opposite strands of a target DNA (paragraph [0066]). The DNA-targeting RNAs of Fraser read on the guide RNAs of Fraser, which are disclosed to comprise a crRNA spacer sequence that is complementary to a target nucleic acid sequence (paragraph [0127]).
Given that copending application ‘738 also discloses PEgRNAs, as in the instant application, that Fraser and Komor collectively disclose a method of editing a target genome utilizing said PEgRNAs, that Huang and UniProt disclose the napDNAbp and MMLV-RT sequences of the instant and patented applications, and that Zhang discloses the utility of including an MS2 coat protein and associated binding aptamer thereof in such systems, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to utilize the PEgRNA of copending application ‘738 in the systems collectively disclosed by the prior art, as well as to include an MS2 coat protein and associated binding aptamer thereof to predictably prime edit a double-stranded DNA, as instantly claimed. One would have been motivated to make such a modification in order to receive the expected benefit of practicing prime editing.
This is a provisional nonstatutory double patenting rejection.
Claims 1, 11, 13-16, 82-90, and 92-95 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 192, 330, 333, 340, 341, 347, 348, and 351 of Application No. 18/271,656 (corresponds to US 2025/0327045 A1; as cited in the IDS filed 07/15/2026) in view of WO 2018/049168 A1 (hereinafter Fraser; as cited in the IDS filed 08/24/2023), Komor et al., 2017 (hereinafter Komor; as cited in the IDS filed 08/24/2023), US 2016/0289673 A1 (hereinafter Huang), Ohtsubo et al., 2017 (hereinafter Ohtsubo), Yin et al., 2018 (hereinafter Yin), UniProt Entry P03355 (hereinafter UniProt; first entered 07/21/1986), and WO 2019/005884 (hereinafter Zhang; as cited in the IDS filed 08/24/2023).
MPEP 804 II B 1 states:
The specification can be used as a dictionary to learn the meaning of a term in the patent claim. Toro Co. v. White Consol. Indus., Inc., 199 F.3d 1295, 1299, 53 USPQ2d 1065, 1067 (Fed. Cir. 1999)… Further, those portions of the specification which provide support for the patent claims may also be examined and considered when addressing the issue of whether a claim in the application defines an obvious variation of an invention claimed in the patent. In re Vogel, 422 F.2d 438, 441-42, 164 USPQ 619, 622 (CCPA 1970).
The following rejections are in view of the decision of the Court of Appeals for the Federal Circuit in Pfizer Inc, v Teva pharmaceuticals USA Inc., 86 USPQ2d 1001, at page 1008 (March 2008), which indicates that there is no patentable distinction between claims to a product and a method of using that product disclosed in the specification of the application and that the preclusion of such a double patenting rejection under 35 USC 121 does not apply where the present application is other than a divisional application of the patent application containing such patentably indistinct claims.
Copending claim 192 is drawn to a prime editor comprising a napDNAbp and a DNA polymerase, wherein the napDNAbp is a Cas9 nickase, as recited at instant claims 1 and 11. Copending claim 347 further recites that the prime editor system claimed therein further comprises a prime editing guide RNA, as recited at instant claim 1. Copending claim 351 is drawn to a method of editing a nucleic acid molecule, said method comprising contacting the nucleic acid molecule with the prime editor system of claim 347 (as set forth above).
With regard to claim 13, which recites “the napDNAbp [of the method of claim 1] comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 137,” as set forth above, Fraser discloses that a Cas9 nickase may be used in the editing methods disclosed therein. However, Fraser is silent as to the sequence of said Cas9 nickase. This deficiency is cured by Huang.
Huang discloses that an exemplary nicking Cas9 protein is Cas9 H840A, which corresponds to SEQ ID NO: 6 taught therein (paragraphs [0077] and [0080]). As shown in the alignment of the Appendix, SEQ ID NO: 6 of Huang comprises 100% identity to instant SEQ ID NO: 137. This H840A mutation is also recited at copending claim 333.
With regard to claim 14, which recites “the polymerase [of the method of claim 1] is a reverse transcriptase,” as set forth above, Fraser discloses coordinated actions of a Cas nickase and a reverse transcriptase (paragraphs [0009], [0106], [0147], [0148], and [0154]). This is also recited at copending claim 340.
With regard to claim 15, which recites “the reverse transcriptase [of the method of claim 14] comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 139,” as set forth above, Fraser discloses that a reverse transcriptase may be used in the editing methods disclosed therein. However, Fraser is silent as to the sequence of said reverse transcriptase. This deficiency is cured by Ohtsubo, Yin, and Uniprot, as set forth below.
Ohtsubo teaches that the Moloney murine leukemia virus (MMLV) reverse transcriptase (MMLV-RT) is widely used for cDNA synthesis (i.e. RNA to DNA) but also has DNA polymerase activity when using DNA as a template (page 1, paragraph 3 of the introduction). Per Yin, adding DNA residues to CRISPR guide RNAs significantly reduces off-target genome editing and reduces the associated cost (abstract). Therefore, prior to the effective filing date of the instant invention, one of ordinary skill in the art would have been motivated to generate a guide RNA per the teachings of Yin that comprises DNA residues. Therefore, the reverse transcriptase disclosed in Fraser must be capable of synthesizing DNA from both DNA and RNA in order to utilize both chimeric guide RNAs and standard guide RNAs efficiently. As set forth above, Ohtsubo teaches that MMLV-RT is uniquely suited to both RNA to DNA synthesis and DNA to DNA synthesis. Accordingly, one of ordinary skill in the art would have been motivated to utilize MMLV-RT in the methods of Fraser. UniProt teaches the sequence of MMLV-RT, which comprises 98.8% sequence identity to instant SEQ ID NO: 139, as shown in the alignment of the Appendix. Copending claim 41 also recite that the reverse transcriptase claimed therein is a retroviral reverse transcriptase, such as that of the instant claim set.
With regard to claim 16, which recites “the DNA extension arm [of the method of claim 1] is at the 3’ end of the PEgRNA,” Fraser further discloses that the retron-guide RNA cassette taught therein may be structured such that the gRNA coding region is located 3’ or 5’ of the retron (paragraphs [0010] and [0102]). This is also recited at copending claim 2.
With regard to claim 82, which recites “the prime editor [of the method of claim 1] is a fusion protein,” as set forth above, Fraser discloses coordinated actions of a Cas nickase and a reverse transcriptase for purposes of editing a nucleic acid sequence. However, Fraser does not explicitly disclose fusion of the Cas nickase and the reverse transcriptase taught therein. This deficiency is cured by Komor. Komor discloses base editors, which comprise a nickase Cas9 such as nSpCas9 and nSaCas9 fused to a base editing enzyme such as APOBEC1 (Figure 2). While the disclosure of Fraser and Komor differ in their effects, both disclosures are drawn to coordinated actions of a Cas nickase and an effector protein for purposes of editing a targeted nucleic acid. Therefore, based on these disclosures, one of ordinary skill in the art would have been motivated, prior to the effective filing date of the instant application, to apply the principles disclosed in Komor to the methods of Fraser such that the Cas nickase disclosed therein is fused to the reverse transcriptase disclosed therein. This is also recited at copending claim 330.
With regard to claim 83, which recites “the napDNAbp [of the method of claim 82] is N-terminal of the polymerase in the fusion protein,” as set forth above, Fraser and Komor collectively disclose a fusion protein comprising a Cas nickase and a reverse transcriptase. However, neither specifically teaches the architecture of said fusion protein. Given that the fusion protein collectively disclosed by Fraser and Komor comprises only two protein components, there are only two possible arrangements: one in which the Cas nickase is N-terminal of the reverse transcriptase in the fusion protein, and one in which the Cas nickase is C-terminal of the reverse transcriptase in the fusion protein. Per MPEP § 2144.04(VI)(C), it is not inventive to rearrange parts. Given that there are only a small, finite number of possible arrangements, it is not considered inventive to merely rearrange these components.
With regard to claim 84, which recites “the prime editor [of the method of claim 83] further comprises a peptide linker between the napDNAbp and the polymerase,” as set forth above, Fraser and Komor collectively disclose the method of claim 83. However, neither Fraser nor Komor disclose the peptide linker of instant claim 84. This deficiency is cured by Zhang.
Zhang discloses methods and compositions for targeting and editing nucleic acids, particularly RNA targeted and edited via a Cas13 fusion protein comprising a Cas13 fused to an editing enzyme such as ADAR (abstract; paragraph [0047]; Figure 2). While the methods of Zhang are drawn to RNA editing and not DNA editing, Zhang nonetheless discloses coordinated activity of a Cas enzyme and an editing enzyme, as in the instant application, Fraser, and Komor. Zhang further discloses that the fusion proteins taught therein may be linked via a peptide linker (paragraphs [0200] and [0201]). Thus, Zhang establishes that Cas proteins may be fused to effector proteins via a peptide linker, as instantly claimed.
With regard to claim 85, which recites “the polymerase [of the method of claim 1] is not connected to the napDNAbp in a fusion protein,” as set forth above, Fraser discloses coordinated actions of a Cas9 nickase and a reverse transcriptase for purposes of editing a nucleic acid sequence. Fraser further discloses that the reverse transcriptase and Cas effector disclosed therein may be provided separately (paragraphs [0026] and [0027]).
With regard to claim 86, which recites “the PEgRNA further comprises an RNA-binding protein recruitment domain,” as set forth above, the retron-guide cassette of Fraser reads on the instantly claimed PEgRNA. Zhang further discloses that the guide molecule taught therein comprises an aptamer sequence capable of binding to an RNA-binding adaptor protein such as MS2 (paragraphs [0011], [0315], and [0316), which reads on the instantly claimed RNA-binding protein recruitment domain.
With regard to claim 87, which recites “the RNA-binding protein recruitment domain [of the method of claim 86] is an MS2 aptamer,” as set forth above, Zhang further discloses that the guide molecule taught therein comprises an aptamer sequence capable of binding to an RNA-binding adaptor protein such as MS2 (paragraphs [0011], [0315], and [0316).
With regard to claim 88, which recites “the polymerase [of the method of claim 85] is fused to an RNA-binding protein domain,” Zhang further discloses that the adenosine deaminase effector taught therein may be fused to RNA-binding protein MS2 coat protein (paragraphs [0315] and [0316]). While the adenosine deaminase effector of Zhang does not read on the instantly claimed polymerase, the disclosure of Zhang is applied to establish that principles of coordinating activity of a Cas protein and an editing enzyme, as in the instant application, Fraser, and Komor. Therefore, Zhang teaches that the effector/editing enzyme taught therein may be fused to an RNA-binding protein such as MS2 coat protein for purposes of implementing orthogonal gene editing (paragraphs [0315], [0316], and [0325]).
With regard to claim 89, which recites “the polymerase [of the method of claim 86] is fused to an MS2cp domain,” as set forth above, Zhang further discloses that the adenosine deaminase effector taught therein may be fused to RNA-binding protein MS2 coat protein for purposes of implementing orthogonal gene editing (paragraphs [0315], [0316], and [0325]).
With regard to claim 90, which recites “the double stranded DNA [of the method of claim 1] is in a cell,” Fraser discloses that the methods taught therein may be practiced within a genome of a host cell, wherein the host cell is transformed with a vector carrying the machinery set forth above and is subsequently edited (paragraph [0024]).
With regard to claim 92, which recites “the method of claim 90, further comprising delivering an mRNA encoding the prime editor into the cell,” as set forth above, Fraser and Komor collectively disclose the instantly claimed prime editor. While Fraser discloses that the reverse transcriptase and Cas effector taught therein may be expressed from a vector or plasmid (paragraphs [0026], [0027], and [0145]), Fraser does not explicitly disclose that these components are delivered as an mRNA. This deficiency is cured by Zhang, which discloses that the editing compositions disclosed therein (including Cas-enzyme fusions) may be delivered as mRNA molecules encoding all of the components thereof (paragraphs [0318] and [0319]).
With regard to claim 93, which recites “the mRNA and the pegRNA [of the method of claim 92] are delivered via a lipid nanoparticle,” Zhang further discloses that the mRNA encoding the components of the system taught therein (and set forth above) may be delivered via one or more lipid nanoparticles (paragraphs [0318] and [0319]).
With regard to claim 94, which recites “the method of claim 1, further comprising contacting the double stranded DNA with a complex comprising the prime editor and a nicking guide RNA (ngRNA),” Fraser further discloses that providing at least two DNA-targeting RNAs targeting opposite DNA strands facilitates a double-nicked double-strand break that favors HDR and decreases the frequency of insertion/deletion mutations at off-target DNA sites (paragraph [0143]). Per Fraser, two nickases of the invention disclosed therein may be utilized to create two single-stranded breaks on opposite strands of a target DNA, as set forth above (paragraph [0066]). This is also recited at copending claim 348.
With regard to claim 95, which recites “the ngRNA [of the method of claim 94] comprises a[n] ngRNA spacer sequence capable of targeting the napDNAbp to the non-target strand of the double-stranded DNA sequence,” as set forth above, Fraser further discloses that providing at least two DNA-targeting RNAs targeting opposite DNA strands facilitates a double-nicked double-strand break that favors HDR and decreases the frequency of insertion/deletion mutations at off-target DNA sites (paragraph [0143]). Per Fraser, two nickases of the invention disclosed therein may be utilized to create two single-stranded breaks on opposite strands of a target DNA (paragraph [0066]). The DNA-targeting RNAs of Fraser read on the guide RNAs of Fraser, which are disclosed to comprise a crRNA spacer sequence that is complementary to a target nucleic acid sequence (paragraph [0127]).
Given that copending application ‘656 also discloses PEgRNAs, as in the instant application, that Fraser and Komor collectively disclose a method of editing a target genome utilizing said PEgRNAs, that Huang and UniProt disclose the napDNAbp and MMLV-RT sequences of the instant and patented applications, and that Zhang discloses the utility of including an MS2 coat protein and associated binding aptamer thereof in such systems, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to utilize the PEgRNA of copending application ‘656 in the systems collectively disclosed by the prior art, as well as to include an MS2 coat protein and associated binding aptamer thereof to predictably prime edit a double-stranded DNA, as instantly claimed. One would have been motivated to make such a modification in order to receive the expected benefit of practicing prime editing.
This is a provisional nonstatutory double patenting rejection.
Claims 1, 11, 13-16, 82-90, and 92-95 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 25, 101, and 117 of Application No. 18/712,922 (corresponds to US 2025/0297246 A1) in view of WO 2018/049168 A1 (hereinafter Fraser; as cited in the IDS filed 08/24/2023), Komor et al., 2017 (hereinafter Komor; as cited in the IDS filed 08/24/2023), US 2016/0289673 A1 (hereinafter Huang), Ohtsubo et al., 2017 (hereinafter Ohtsubo), Yin et al., 2018 (hereinafter Yin), UniProt Entry P03355 (hereinafter UniProt; first entered 07/21/1986), and WO 2019/005884 (hereinafter Zhang; as cited in the IDS filed 08/24/2023).
MPEP 804 II B 1 states:
The specification can be used as a dictionary to learn the meaning of a term in the patent claim. Toro Co. v. White Consol. Indus., Inc., 199 F.3d 1295, 1299, 53 USPQ2d 1065, 1067 (Fed. Cir. 1999)… Further, those portions of the specification which provide support for the patent claims may also be examined and considered when addressing the issue of whether a claim in the application defines an obvious variation of an invention claimed in the patent. In re Vogel, 422 F.2d 438, 441-42, 164 USPQ 619, 622 (CCPA 1970).
The following rejections are in view of the decision of the Court of Appeals for the Federal Circuit in Pfizer Inc, v Teva pharmaceuticals USA Inc., 86 USPQ2d 1001, at page 1008 (March 2008), which indicates that there is no patentable distinction between claims to a product and a method of using that product disclosed in the specification of the application and that the preclusion of such a double patenting rejection under 35 USC 121 does not apply where the present application is other than a divisional application of the patent application containing such patentably indistinct claims.
Copending claim 25 is also drawn to a prime editing guide RNA comprising a spacer, and extension arm, and a guide RNA core. Copending claim 101 is drawn to a prime editing system comprising said prime editing guide RNA, as well as a prime editor comprising a Cas9 protein and a reverse transcriptase, wherein the Cas9 protein comprises a mutation in an HNH domain. Finally, copending claim 117 is drawn to a method for editing a double stranded target DNA, said method comprising contacting the target DNA with the PEgRNA set forth above, as well as a prime editor comprising a Cas9 nickase and a reverse transcriptase.
The prime editing guide RNA of the copending application reads on the prime editing guide RNA of the instant application as recited at instant claim 1. Furthermore, the prime editor recited in the copending application reads on the prime editor of the instant application, as recited at instant claims 1 and 11. The method recited at copending claim 117 reads on the method of instant claim 1.
Regarding the additional limitations of the instant application, the prior art teaches these limitations, as set forth above.
With regard to claim 13, which recites “the napDNAbp [of the method of claim 1] comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 137,” as set forth above, Fraser discloses that a Cas9 nickase may be used in the editing methods disclosed therein. However, Fraser is silent as to the sequence of said Cas9 nickase. This deficiency is cured by Huang.
Huang discloses that an exemplary nicking Cas9 protein is Cas9 H840A, which corresponds to SEQ ID NO: 6 taught therein (paragraphs [0077] and [0080]). As shown in the alignment of the Appendix, SEQ ID NO: 6 of Huang comprises 100% identity to instant SEQ ID NO: 137. This H840A mutation reads on the mutation recited at copending claim 101.
With regard to claim 14, which recites “the polymerase [of the method of claim 1] is a reverse transcriptase,” as set forth above, Fraser discloses coordinated actions of a Cas nickase and a reverse transcriptase (paragraphs [0009], [0106], [0147], [0148], and [0154]). This is also recited at copending claim 340.
With regard to claim 15, which recites “the reverse transcriptase [of the method of claim 14] comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 139,” as set forth above, Fraser discloses that a reverse transcriptase may be used in the editing methods disclosed therein. However, Fraser is silent as to the sequence of said reverse transcriptase. This deficiency is cured by Ohtsubo, Yin, and Uniprot, as set forth below.
Ohtsubo teaches that the Moloney murine leukemia virus (MMLV) reverse transcriptase (MMLV-RT) is widely used for cDNA synthesis (i.e. RNA to DNA) but also has DNA polymerase activity when using DNA as a template (page 1, paragraph 3 of the introduction). Per Yin, adding DNA residues to CRISPR guide RNAs significantly reduces off-target genome editing and reduces the associated cost (abstract). Therefore, prior to the effective filing date of the instant invention, one of ordinary skill in the art would have been motivated to generate a guide RNA per the teachings of Yin that comprises DNA residues. Therefore, the reverse transcriptase disclosed in Fraser must be capable of synthesizing DNA from both DNA and RNA in order to utilize both chimeric guide RNAs and standard guide RNAs efficiently. As set forth above, Ohtsubo teaches that MMLV-RT is uniquely suited to both RNA to DNA synthesis and DNA to DNA synthesis. Accordingly, one of ordinary skill in the art would have been motivated to utilize MMLV-RT in the methods of Fraser. UniProt teaches the sequence of MMLV-RT, which comprises 98.8% sequence identity to instant SEQ ID NO: 139, as shown in the alignment of the Appendix.
With regard to claim 16, which recites “the DNA extension arm [of the method of claim 1] is at the 3’ end of the PEgRNA,” Fraser further discloses that the retron-guide RNA cassette taught therein may be structured such that the gRNA coding region is located 3’ or 5’ of the retron (paragraphs [0010] and [0102]).
With regard to claim 82, which recites “the prime editor [of the method of claim 1] is a fusion protein,” as set forth above, Fraser discloses coordinated actions of a Cas nickase and a reverse transcriptase for purposes of editing a nucleic acid sequence. However, Fraser does not explicitly disclose fusion of the Cas nickase and the reverse transcriptase taught therein. This deficiency is cured by Komor. Komor discloses base editors, which comprise a nickase Cas9 such as nSpCas9 and nSaCas9 fused to a base editing enzyme such as APOBEC1 (Figure 2). While the disclosure of Fraser and Komor differ in their effects, both disclosures are drawn to coordinated actions of a Cas nickase and an effector protein for purposes of editing a targeted nucleic acid. Therefore, based on these disclosures, one of ordinary skill in the art would have been motivated, prior to the effective filing date of the instant application, to apply the principles disclosed in Komor to the methods of Fraser such that the Cas nickase disclosed therein is fused to the reverse transcriptase disclosed therein.
With regard to claim 83, which recites “the napDNAbp [of the method of claim 82] is N-terminal of the polymerase in the fusion protein,” as set forth above, Fraser and Komor collectively disclose a fusion protein comprising a Cas nickase and a reverse transcriptase. However, neither specifically teaches the architecture of said fusion protein. Given that the fusion protein collectively disclosed by Fraser and Komor comprises only two protein components, there are only two possible arrangements: one in which the Cas nickase is N-terminal of the reverse transcriptase in the fusion protein, and one in which the Cas nickase is C-terminal of the reverse transcriptase in the fusion protein. Per MPEP § 2144.04(VI)(C), it is not inventive to rearrange parts. Given that there are only a small, finite number of possible arrangements, it is not considered inventive to merely rearrange these components.
With regard to claim 84, which recites “the prime editor [of the method of claim 83] further comprises a peptide linker between the napDNAbp and the polymerase,” as set forth above, Fraser and Komor collectively disclose the method of claim 83. However, neither Fraser nor Komor disclose the peptide linker of instant claim 84. This deficiency is cured by Zhang.
Zhang discloses methods and compositions for targeting and editing nucleic acids, particularly RNA targeted and edited via a Cas13 fusion protein comprising a Cas13 fused to an editing enzyme such as ADAR (abstract; paragraph [0047]; Figure 2). While the methods of Zhang are drawn to RNA editing and not DNA editing, Zhang nonetheless discloses coordinated activity of a Cas enzyme and an editing enzyme, as in the instant application, Fraser, and Komor. Zhang further discloses that the fusion proteins taught therein may be linked via a peptide linker (paragraphs [0200] and [0201]). Thus, Zhang establishes that Cas proteins may be fused to effector proteins via a peptide linker, as instantly claimed.
With regard to claim 85, which recites “the polymerase [of the method of claim 1] is not connected to the napDNAbp in a fusion protein,” as set forth above, Fraser discloses coordinated actions of a Cas9 nickase and a reverse transcriptase for purposes of editing a nucleic acid sequence. Fraser further discloses that the reverse transcriptase and Cas effector disclosed therein may be provided separately (paragraphs [0026] and [0027]).
With regard to claim 86, which recites “the PEgRNA further comprises an RNA-binding protein recruitment domain,” as set forth above, the retron-guide cassette of Fraser reads on the instantly claimed PEgRNA. Zhang further discloses that the guide molecule taught therein comprises an aptamer sequence capable of binding to an RNA-binding adaptor protein such as MS2 (paragraphs [0011], [0315], and [0316), which reads on the instantly claimed RNA-binding protein recruitment domain.
With regard to claim 87, which recites “the RNA-binding protein recruitment domain [of the method of claim 86] is an MS2 aptamer,” as set forth above, Zhang further discloses that the guide molecule taught therein comprises an aptamer sequence capable of binding to an RNA-binding adaptor protein such as MS2 (paragraphs [0011], [0315], and [0316).
With regard to claim 88, which recites “the polymerase [of the method of claim 85] is fused to an RNA-binding protein domain,” Zhang further discloses that the adenosine deaminase effector taught therein may be fused to RNA-binding protein MS2 coat protein (paragraphs [0315] and [0316]). While the adenosine deaminase effector of Zhang does not read on the instantly claimed polymerase, the disclosure of Zhang is applied to establish that principles of coordinating activity of a Cas protein and an editing enzyme, as in the instant application, Fraser, and Komor. Therefore, Zhang teaches that the effector/editing enzyme taught therein may be fused to an RNA-binding protein such as MS2 coat protein for purposes of implementing orthogonal gene editing (paragraphs [0315], [0316], and [0325]).
With regard to claim 89, which recites “the polymerase [of the method of claim 86] is fused to an MS2cp domain,” as set forth above, Zhang further discloses that the adenosine deaminase effector taught therein may be fused to RNA-binding protein MS2 coat protein for purposes of implementing orthogonal gene editing (paragraphs [0315], [0316], and [0325]).
With regard to claim 90, which recites “the double stranded DNA [of the method of claim 1] is in a cell,” Fraser discloses that the methods taught therein may be practiced within a genome of a host cell, wherein the host cell is transformed with a vector carrying the machinery set forth above and is subsequently edited (paragraph [0024]).
With regard to claim 92, which recites “the method of claim 90, further comprising delivering an mRNA encoding the prime editor into the cell,” as set forth above, Fraser and Komor collectively disclose the instantly claimed prime editor. While Fraser discloses that the reverse transcriptase and Cas effector taught therein may be expressed from a vector or plasmid (paragraphs [0026], [0027], and [0145]), Fraser does not explicitly disclose that these components are delivered as an mRNA. This deficiency is cured by Zhang, which discloses that the editing compositions disclosed therein (including Cas-enzyme fusions) may be delivered as mRNA molecules encoding all of the components thereof (paragraphs [0318] and [0319]).
With regard to claim 93, which recites “the mRNA and the pegRNA [of the method of claim 92] are delivered via a lipid nanoparticle,” Zhang further discloses that the mRNA encoding the components of the system taught therein (and set forth above) may be delivered via one or more lipid nanoparticles (paragraphs [0318] and [0319]).
With regard to claim 94, which recites “the method of claim 1, further comprising contacting the double stranded DNA with a complex comprising the prime editor and a nicking guide RNA (ngRNA),” Fraser further discloses that providing at least two DNA-targeting RNAs targeting opposite DNA strands facilitates a double-nicked double-strand break that favors HDR and decreases the frequency of insertion/deletion mutations at off-target DNA sites (paragraph [0143]). Per Fraser, two nickases of the invention disclosed therein may be utilized to create two single-stranded breaks on opposite strands of a target DNA, as set forth above (paragraph [0066]).
With regard to claim 95, which recites “the ngRNA [of the method of claim 94] comprises a[n] ngRNA spacer sequence capable of targeting the napDNAbp to the non-target strand of the double-stranded DNA sequence,” as set forth above, Fraser further discloses that providing at least two DNA-targeting RNAs targeting opposite DNA strands facilitates a double-nicked double-strand break that favors HDR and decreases the frequency of insertion/deletion mutations at off-target DNA sites (paragraph [0143]). Per Fraser, two nickases of the invention disclosed therein may be utilized to create two single-stranded breaks on opposite strands of a target DNA (paragraph [0066]). The DNA-targeting RNAs of Fraser read on the guide RNAs of Fraser, which are disclosed to comprise a crRNA spacer sequence that is complementary to a target nucleic acid sequence (paragraph [0127]).
Given that copending application ‘922 also discloses PEgRNAs, as in the instant application, that Fraser and Komor collectively disclose a method of editing a target genome utilizing said PEgRNAs, that Huang and UniProt disclose the napDNAbp and MMLV-RT sequences of the instant and patented applications, and that Zhang discloses the utility of including an MS2 coat protein and associated binding aptamer thereof in such systems, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to utilize the PEgRNA of copending application ‘922 in the systems collectively disclosed by the prior art, as well as to include an MS2 coat protein and associated binding aptamer thereof to predictably prime edit a double-stranded DNA, as instantly claimed. One would have been motivated to make such a modification in order to receive the expected benefit of practicing prime editing.
This is a provisional nonstatutory double patenting rejection.
Claims 1, 11, 13-16, 82-90, and 92-95 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 140, 146, 148, and 154 of Application No. 18/903,447 (corresponds to US 2025/0243474 A1) in view of WO 2018/049168 A1 (hereinafter Fraser; as cited in the IDS filed 08/24/2023), Komor et al., 2017 (hereinafter Komor; as cited in the IDS filed 08/24/2023), US 2016/0289673 A1 (hereinafter Huang), Ohtsubo et al., 2017 (hereinafter Ohtsubo), Yin et al., 2018 (hereinafter Yin), UniProt Entry P03355 (hereinafter UniProt; first entered 07/21/1986), and WO 2019/005884 (hereinafter Zhang; as cited in the IDS filed 08/24/2023).
MPEP 804 II B 1 states:
The specification can be used as a dictionary to learn the meaning of a term in the patent claim. Toro Co. v. White Consol. Indus., Inc., 199 F.3d 1295, 1299, 53 USPQ2d 1065, 1067 (Fed. Cir. 1999)… Further, those portions of the specification which provide support for the patent claims may also be examined and considered when addressing the issue of whether a claim in the application defines an obvious variation of an invention claimed in the patent. In re Vogel, 422 F.2d 438, 441-42, 164 USPQ 619, 622 (CCPA 1970).
The following rejections are in view of the decision of the Court of Appeals for the Federal Circuit in Pfizer Inc, v Teva pharmaceuticals USA Inc., 86 USPQ2d 1001, at page 1008 (March 2008), which indicates that there is no patentable distinction between claims to a product and a method of using that product disclosed in the specification of the application and that the preclusion of such a double patenting rejection under 35 USC 121 does not apply where the present application is other than a divisional application of the patent application containing such patentably indistinct claims.
Copending claim 140 is also drawn to a prime editing guide RNA comprising a spacer, and extension arm, and a guide RNA core. Copending claim 146 is drawn to a prime editing system comprising said prime editing guide RNA, as well as a prime editor comprising a Cas9 nickase (as recited at instant claim 11) and a reverse transcriptase (as recited at instant claim 14). Finally, copending claim 148 is drawn to a method for editing a double stranded target DNA, said method comprising contacting the target DNA with the PEgRNA set forth above, as well as a prime editor comprising a Cas9 nickase and a reverse transcriptase.
The prime editing guide RNA of the copending application reads on the prime editing guide RNA of the instant application as recited at instant claim 1. Furthermore, the prime editor recited in the copending application reads on the prime editor of the instant application, as recited at instant claims 1, 11, and 14. The method recited at copending claim 117 reads on the method of instant claim 1.
Regarding the additional limitations of the instant application, the prior art teaches these limitations, as set forth above.
With regard to claim 13, which recites “the napDNAbp [of the method of claim 1] comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 137,” as set forth above, Fraser discloses that a Cas9 nickase may be used in the editing methods disclosed therein. However, Fraser is silent as to the sequence of said Cas9 nickase. This deficiency is cured by Huang.
Huang discloses that an exemplary nicking Cas9 protein is Cas9 H840A, which corresponds to SEQ ID NO: 6 taught therein (paragraphs [0077] and [0080]). As shown in the alignment of the Appendix, SEQ ID NO: 6 of Huang comprises 100% identity to instant SEQ ID NO: 137.
With regard to claim 14, which recites “the polymerase [of the method of claim 1] is a reverse transcriptase,” as set forth above, Fraser discloses coordinated actions of a Cas nickase and a reverse transcriptase (paragraphs [0009], [0106], [0147], [0148], and [0154]).
With regard to claim 15, which recites “the reverse transcriptase [of the method of claim 14] comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 139,” as set forth above, Fraser discloses that a reverse transcriptase may be used in the editing methods disclosed therein. However, Fraser is silent as to the sequence of said reverse transcriptase. This deficiency is cured by Ohtsubo, Yin, and Uniprot, as set forth below.
Ohtsubo teaches that the Moloney murine leukemia virus (MMLV) reverse transcriptase (MMLV-RT) is widely used for cDNA synthesis (i.e. RNA to DNA) but also has DNA polymerase activity when using DNA as a template (page 1, paragraph 3 of the introduction). Per Yin, adding DNA residues to CRISPR guide RNAs significantly reduces off-target genome editing and reduces the associated cost (abstract). Therefore, prior to the effective filing date of the instant invention, one of ordinary skill in the art would have been motivated to generate a guide RNA per the teachings of Yin that comprises DNA residues. Therefore, the reverse transcriptase disclosed in Fraser must be capable of synthesizing DNA from both DNA and RNA in order to utilize both chimeric guide RNAs and standard guide RNAs efficiently. As set forth above, Ohtsubo teaches that MMLV-RT is uniquely suited to both RNA to DNA synthesis and DNA to DNA synthesis. Accordingly, one of ordinary skill in the art would have been motivated to utilize MMLV-RT in the methods of Fraser. UniProt teaches the sequence of MMLV-RT, which comprises 98.8% sequence identity to instant SEQ ID NO: 139, as shown in the alignment of the Appendix.
With regard to claim 16, which recites “the DNA extension arm [of the method of claim 1] is at the 3’ end of the PEgRNA,” Fraser further discloses that the retron-guide RNA cassette taught therein may be structured such that the gRNA coding region is located 3’ or 5’ of the retron (paragraphs [0010] and [0102]).
With regard to claim 82, which recites “the prime editor [of the method of claim 1] is a fusion protein,” as set forth above, Fraser discloses coordinated actions of a Cas nickase and a reverse transcriptase for purposes of editing a nucleic acid sequence. However, Fraser does not explicitly disclose fusion of the Cas nickase and the reverse transcriptase taught therein. This deficiency is cured by Komor. Komor discloses base editors, which comprise a nickase Cas9 such as nSpCas9 and nSaCas9 fused to a base editing enzyme such as APOBEC1 (Figure 2). While the disclosure of Fraser and Komor differ in their effects, both disclosures are drawn to coordinated actions of a Cas nickase and an effector protein for purposes of editing a targeted nucleic acid. Therefore, based on these disclosures, one of ordinary skill in the art would have been motivated, prior to the effective filing date of the instant application, to apply the principles disclosed in Komor to the methods of Fraser such that the Cas nickase disclosed therein is fused to the reverse transcriptase disclosed therein.
With regard to claim 83, which recites “the napDNAbp [of the method of claim 82] is N-terminal of the polymerase in the fusion protein,” as set forth above, Fraser and Komor collectively disclose a fusion protein comprising a Cas nickase and a reverse transcriptase. However, neither specifically teaches the architecture of said fusion protein. Given that the fusion protein collectively disclosed by Fraser and Komor comprises only two protein components, there are only two possible arrangements: one in which the Cas nickase is N-terminal of the reverse transcriptase in the fusion protein, and one in which the Cas nickase is C-terminal of the reverse transcriptase in the fusion protein. Per MPEP § 2144.04(VI)(C), it is not inventive to rearrange parts. Given that there are only a small, finite number of possible arrangements, it is not considered inventive to merely rearrange these components.
With regard to claim 84, which recites “the prime editor [of the method of claim 83] further comprises a peptide linker between the napDNAbp and the polymerase,” as set forth above, Fraser and Komor collectively disclose the method of claim 83. However, neither Fraser nor Komor disclose the peptide linker of instant claim 84. This deficiency is cured by Zhang.
Zhang discloses methods and compositions for targeting and editing nucleic acids, particularly RNA targeted and edited via a Cas13 fusion protein comprising a Cas13 fused to an editing enzyme such as ADAR (abstract; paragraph [0047]; Figure 2). While the methods of Zhang are drawn to RNA editing and not DNA editing, Zhang nonetheless discloses coordinated activity of a Cas enzyme and an editing enzyme, as in the instant application, Fraser, and Komor. Zhang further discloses that the fusion proteins taught therein may be linked via a peptide linker (paragraphs [0200] and [0201]). Thus, Zhang establishes that Cas proteins may be fused to effector proteins via a peptide linker, as instantly claimed.
With regard to claim 85, which recites “the polymerase [of the method of claim 1] is not connected to the napDNAbp in a fusion protein,” as set forth above, Fraser discloses coordinated actions of a Cas9 nickase and a reverse transcriptase for purposes of editing a nucleic acid sequence. Fraser further discloses that the reverse transcriptase and Cas effector disclosed therein may be provided separately (paragraphs [0026] and [0027]).
With regard to claim 86, which recites “the PEgRNA further comprises an RNA-binding protein recruitment domain,” as set forth above, the retron-guide cassette of Fraser reads on the instantly claimed PEgRNA. Zhang further discloses that the guide molecule taught therein comprises an aptamer sequence capable of binding to an RNA-binding adaptor protein such as MS2 (paragraphs [0011], [0315], and [0316), which reads on the instantly claimed RNA-binding protein recruitment domain.
With regard to claim 87, which recites “the RNA-binding protein recruitment domain [of the method of claim 86] is an MS2 aptamer,” as set forth above, Zhang further discloses that the guide molecule taught therein comprises an aptamer sequence capable of binding to an RNA-binding adaptor protein such as MS2 (paragraphs [0011], [0315], and [0316).
With regard to claim 88, which recites “the polymerase [of the method of claim 85] is fused to an RNA-binding protein domain,” Zhang further discloses that the adenosine deaminase effector taught therein may be fused to RNA-binding protein MS2 coat protein (paragraphs [0315] and [0316]). While the adenosine deaminase effector of Zhang does not read on the instantly claimed polymerase, the disclosure of Zhang is applied to establish that principles of coordinating activity of a Cas protein and an editing enzyme, as in the instant application, Fraser, and Komor. Therefore, Zhang teaches that the effector/editing enzyme taught therein may be fused to an RNA-binding protein such as MS2 coat protein for purposes of implementing orthogonal gene editing (paragraphs [0315], [0316], and [0325]).
With regard to claim 89, which recites “the polymerase [of the method of claim 86] is fused to an MS2cp domain,” as set forth above, Zhang further discloses that the adenosine deaminase effector taught therein may be fused to RNA-binding protein MS2 coat protein for purposes of implementing orthogonal gene editing (paragraphs [0315], [0316], and [0325]).
With regard to claim 90, which recites “the double stranded DNA [of the method of claim 1] is in a cell,” Fraser discloses that the methods taught therein may be practiced within a genome of a host cell, wherein the host cell is transformed with a vector carrying the machinery set forth above and is subsequently edited (paragraph [0024]).
With regard to claim 92, which recites “the method of claim 90, further comprising delivering an mRNA encoding the prime editor into the cell,” as set forth above, Fraser and Komor collectively disclose the instantly claimed prime editor. While Fraser discloses that the reverse transcriptase and Cas effector taught therein may be expressed from a vector or plasmid (paragraphs [0026], [0027], and [0145]), Fraser does not explicitly disclose that these components are delivered as an mRNA. This deficiency is cured by Zhang, which discloses that the editing compositions disclosed therein (including Cas-enzyme fusions) may be delivered as mRNA molecules encoding all of the components thereof (paragraphs [0318] and [0319]).
With regard to claim 93, which recites “the mRNA and the pegRNA [of the method of claim 92] are delivered via a lipid nanoparticle,” Zhang further discloses that the mRNA encoding the components of the system taught therein (and set forth above) may be delivered via one or more lipid nanoparticles (paragraphs [0318] and [0319]).
With regard to claim 94, which recites “the method of claim 1, further comprising contacting the double stranded DNA with a complex comprising the prime editor and a nicking guide RNA (ngRNA),” Fraser further discloses that providing at least two DNA-targeting RNAs targeting opposite DNA strands facilitates a double-nicked double-strand break that favors HDR and decreases the frequency of insertion/deletion mutations at off-target DNA sites (paragraph [0143]). Per Fraser, two nickases of the invention disclosed therein may be utilized to create two single-stranded breaks on opposite strands of a target DNA, as set forth above (paragraph [0066]).
With regard to claim 95, which recites “the ngRNA [of the method of claim 94] comprises a[n] ngRNA spacer sequence capable of targeting the napDNAbp to the non-target strand of the double-stranded DNA sequence,” as set forth above, Fraser further discloses that providing at least two DNA-targeting RNAs targeting opposite DNA strands facilitates a double-nicked double-strand break that favors HDR and decreases the frequency of insertion/deletion mutations at off-target DNA sites (paragraph [0143]). Per Fraser, two nickases of the invention disclosed therein may be utilized to create two single-stranded breaks on opposite strands of a target DNA (paragraph [0066]). The DNA-targeting RNAs of Fraser read on the guide RNAs of Fraser, which are disclosed to comprise a crRNA spacer sequence that is complementary to a target nucleic acid sequence (paragraph [0127]). The nicking guide RNAs of the instant application are also recited at copending claim 154.
Given that copending application ‘447 also discloses PEgRNAs, as in the instant application, that Fraser and Komor collectively disclose a method of editing a target genome utilizing said PEgRNAs, that Huang and UniProt disclose the napDNAbp and MMLV-RT sequences of the instant and patented applications, and that Zhang discloses the utility of including an MS2 coat protein and associated binding aptamer thereof in such systems, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to utilize the PEgRNA of copending application ‘447 in the systems collectively disclosed by the prior art, as well as to include an MS2 coat protein and associated binding aptamer thereof to predictably prime edit a double-stranded DNA, as instantly claimed. One would have been motivated to make such a modification in order to receive the expected benefit of practicing prime editing.
This is a provisional nonstatutory double patenting rejection.
Claims 1, 11, 13-16, 82-95 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 3, 4, and 9-11 of Application No. 19/368,899 (corresponds to US 2026/0224752 A1) in view of WO 2018/049168 A1 (hereinafter Fraser; as cited in the IDS filed 08/24/2023), Komor et al., 2017 (hereinafter Komor; as cited in the IDS filed 08/24/2023), US 2016/0289673 A1 (hereinafter Huang), Ohtsubo et al., 2017 (hereinafter Ohtsubo), Yin et al., 2018 (hereinafter Yin), UniProt Entry P03355 (hereinafter UniProt; first entered 07/21/1986), and WO 2019/005884 (hereinafter Zhang; as cited in the IDS filed 08/24/2023).
MPEP 804 II B 1 states:
The specification can be used as a dictionary to learn the meaning of a term in the patent claim. Toro Co. v. White Consol. Indus., Inc., 199 F.3d 1295, 1299, 53 USPQ2d 1065, 1067 (Fed. Cir. 1999)… Further, those portions of the specification which provide support for the patent claims may also be examined and considered when addressing the issue of whether a claim in the application defines an obvious variation of an invention claimed in the patent. In re Vogel, 422 F.2d 438, 441-42, 164 USPQ 619, 622 (CCPA 1970).
The following rejections are in view of the decision of the Court of Appeals for the Federal Circuit in Pfizer Inc, v Teva pharmaceuticals USA Inc., 86 USPQ2d 1001, at page 1008 (March 2008), which indicates that there is no patentable distinction between claims to a product and a method of using that product disclosed in the specification of the application and that the preclusion of such a double patenting rejection under 35 USC 121 does not apply where the present application is other than a divisional application of the patent application containing such patentably indistinct claims.
Copending claim 1 is also drawn to a prime editing guide RNA, as well as a prime editor and a nicking guide RNA. Copending claims 3 and 4 further limit the prime editor to comprise a Cas9 nickase. Copending claim 9 is drawn to a method for modifying a target gene in a cell comprising introducing the composition of the copending application.
The prime editing guide RNA of the copending application reads on the prime editing guide RNA of the instant application as recited at instant claim 1.
Regarding the additional limitations of the instant application, the prior art teaches these limitations, as set forth above.
With regard to claim 1, which recites “a method for editing a double-stranded DNA sequence, the method comprising: contacting the double-stranded DNA sequence with a complex comprising a prime editor and a prime editing guide RNA (PEgRNA), wherein the prime editor comprises (i) a nucleic acid programmable DNA binding protein (napDNAbp) capable of nicking a non-target strand of the double stranded DNA sequence to produce a single strand[ed] DNA having a 3’ end and (ii) a polymerase, and wherein the PEgRNA comprises: (i) a spacer sequence capable of targeting the napDNAbp to a target strand of the double stranded DNA sequence, (ii) a gRNA core capable of binding to the napDNAbp, and (iii) a DNA extension arm comprising a DNA synthesis template encoding the desired nucleotide change and a primer binding site capable of hybridizing to the single strand[ed] DNA having the 3’ end,” Fraser discloses methods for high-efficiency genome editing, wherein the method comprises providing retron-guide RNA cassettes (abstract). The retron-guide RNA cassettes of Fraser read on the instantly claimed PEgRNA, as they comprise a retron transcript (i.e. the extension arm of the claim) and a guide RNA molecule comprising a spacer complementary to a first strand and a guide RNA core capable of binding to the Cas napDNAbp taught therein (paragraphs [0003], [0006], [0009], [0010], and [0127]). As set forth above, patent ‘652 also recites PEgRNAs. Fraser explicitly discloses that the retron transcript of the retron-guide RNA cassettes taught therein is capable of self-priming reverse transcription by a reverse transcriptase (paragraph [0020]). The reverse transcriptase of Fraser (see paragraphs [0106], [0147], [0148], and [0154]) reads on the instantly claimed polymerase (see paragraph [0010] of the instant specification), while the Cas nickase disclosed therein (see paragraphs [0066], [0138], [0142], and [0143]) read on the instantly claimed napDNAbp capable of nicking a strand of the double stranded DNA sequence to produce a single stranded DNA having a 3’ end. Thus, Fraser discloses each and every limitation of the instantly claimed method with the exception of specifically nicking the non-target strand. This deficiency is cured by Komor.
While the disclosure of Fraser is drawn to coordinated actions of a Cas nickase (paragraphs [0066], [0138], [0142], and [0143]) and a reverse transcriptase (paragraphs [0020], [0106], [0147], [0148], and [0154]), Cas nickase activity has been coordinated with other effector proteins. Komor discloses base editors, which comprise a nickase Cas9 such as nSpCas9 and nSaCas9 fused to a base editing enzyme such as APOBEC1, wherein the Cas9 nickase nicks the non-edited strand in order to facilitate base editing of the targeted residue in the edited (i.e. targeted) strand (Figure 2). While the disclosure of Fraser and Komor differ in their effects, both disclosures are drawn to coordinated actions of a Cas nickase and an effector protein for purposes of editing a targeted nucleic acid. Therefore, based on these disclosures, one of ordinary skill in the art would have been motivated, prior to the effective filing date of the instant application, to apply the principles disclosed in Komor to the methods of Fraser such that the Cas nickase disclosed therein specifically nicks the non-targeted strand in order to specifically edit the targeted strand, as set forth in greater detail below.
With regard to claim 11, which recites “the napDNAbp [of the method of claim 1] is a Cas9 nickase (nCas9),” as set forth above, Fraser discloses coordinated actions of a Cas nickase and a reverse transcriptase, wherein the Cas nickase is specifically a Cas9 nickase (paragraph [0143]). This is also recited at copending claims 3 and 4.
With regard to claim 13, which recites “the napDNAbp [of the method of claim 1] comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 137,” as set forth above, Fraser discloses that a Cas9 nickase may be used in the editing methods disclosed therein. However, Fraser is silent as to the sequence of said Cas9 nickase. This deficiency is cured by Huang.
Huang discloses that an exemplary nicking Cas9 protein is Cas9 H840A, which corresponds to SEQ ID NO: 6 taught therein (paragraphs [0077] and [0080]). As shown in the alignment of the Appendix, SEQ ID NO: 6 of Huang comprises 100% identity to instant SEQ ID NO: 137.
With regard to claim 14, which recites “the polymerase [of the method of claim 1] is a reverse transcriptase,” as set forth above, Fraser discloses coordinated actions of a Cas nickase and a reverse transcriptase (paragraphs [0009], [0106], [0147], [0148], and [0154]).
With regard to claim 15, which recites “the reverse transcriptase [of the method of claim 14] comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 139,” as set forth above, Fraser discloses that a reverse transcriptase may be used in the editing methods disclosed therein. However, Fraser is silent as to the sequence of said reverse transcriptase. This deficiency is cured by Ohtsubo, Yin, and Uniprot, as set forth below.
Ohtsubo teaches that the Moloney murine leukemia virus (MMLV) reverse transcriptase (MMLV-RT) is widely used for cDNA synthesis (i.e. RNA to DNA) but also has DNA polymerase activity when using DNA as a template (page 1, paragraph 3 of the introduction). Per Yin, adding DNA residues to CRISPR guide RNAs significantly reduces off-target genome editing and reduces the associated cost (abstract). Therefore, prior to the effective filing date of the instant invention, one of ordinary skill in the art would have been motivated to generate a guide RNA per the teachings of Yin that comprises DNA residues. Therefore, the reverse transcriptase disclosed in Fraser must be capable of synthesizing DNA from both DNA and RNA in order to utilize both chimeric guide RNAs and standard guide RNAs efficiently. As set forth above, Ohtsubo teaches that MMLV-RT is uniquely suited to both RNA to DNA synthesis and DNA to DNA synthesis. Accordingly, one of ordinary skill in the art would have been motivated to utilize MMLV-RT in the methods of Fraser. UniProt teaches the sequence of MMLV-RT, which comprises 98.8% sequence identity to instant SEQ ID NO: 139, as shown in the alignment of the Appendix.
With regard to claim 16, which recites “the DNA extension arm [of the method of claim 1] is at the 3’ end of the PEgRNA,” Fraser further discloses that the retron-guide RNA cassette taught therein may be structured such that the gRNA coding region is located 3’ or 5’ of the retron (paragraphs [0010] and [0102]).
With regard to claim 82, which recites “the prime editor [of the method of claim 1] is a fusion protein,” as set forth above, Fraser discloses coordinated actions of a Cas nickase and a reverse transcriptase for purposes of editing a nucleic acid sequence. However, Fraser does not explicitly disclose fusion of the Cas nickase and the reverse transcriptase taught therein. This deficiency is cured by Komor. Komor discloses base editors, which comprise a nickase Cas9 such as nSpCas9 and nSaCas9 fused to a base editing enzyme such as APOBEC1 (Figure 2). While the disclosure of Fraser and Komor differ in their effects, both disclosures are drawn to coordinated actions of a Cas nickase and an effector protein for purposes of editing a targeted nucleic acid. Therefore, based on these disclosures, one of ordinary skill in the art would have been motivated, prior to the effective filing date of the instant application, to apply the principles disclosed in Komor to the methods of Fraser such that the Cas nickase disclosed therein is fused to the reverse transcriptase disclosed therein.
With regard to claim 83, which recites “the napDNAbp [of the method of claim 82] is N-terminal of the polymerase in the fusion protein,” as set forth above, Fraser and Komor collectively disclose a fusion protein comprising a Cas nickase and a reverse transcriptase. However, neither specifically teaches the architecture of said fusion protein. Given that the fusion protein collectively disclosed by Fraser and Komor comprises only two protein components, there are only two possible arrangements: one in which the Cas nickase is N-terminal of the reverse transcriptase in the fusion protein, and one in which the Cas nickase is C-terminal of the reverse transcriptase in the fusion protein. Per MPEP § 2144.04(VI)(C), it is not inventive to rearrange parts. Given that there are only a small, finite number of possible arrangements, it is not considered inventive to merely rearrange these components.
With regard to claim 84, which recites “the prime editor [of the method of claim 83] further comprises a peptide linker between the napDNAbp and the polymerase,” as set forth above, Fraser and Komor collectively disclose the method of claim 83. However, neither Fraser nor Komor disclose the peptide linker of instant claim 84. This deficiency is cured by Zhang.
Zhang discloses methods and compositions for targeting and editing nucleic acids, particularly RNA targeted and edited via a Cas13 fusion protein comprising a Cas13 fused to an editing enzyme such as ADAR (abstract; paragraph [0047]; Figure 2). While the methods of Zhang are drawn to RNA editing and not DNA editing, Zhang nonetheless discloses coordinated activity of a Cas enzyme and an editing enzyme, as in the instant application, Fraser, and Komor. Zhang further discloses that the fusion proteins taught therein may be linked via a peptide linker (paragraphs [0200] and [0201]). Thus, Zhang establishes that Cas proteins may be fused to effector proteins via a peptide linker, as instantly claimed.
With regard to claim 85, which recites “the polymerase [of the method of claim 1] is not connected to the napDNAbp in a fusion protein,” as set forth above, Fraser discloses coordinated actions of a Cas9 nickase and a reverse transcriptase for purposes of editing a nucleic acid sequence. Fraser further discloses that the reverse transcriptase and Cas effector disclosed therein may be provided separately (paragraphs [0026] and [0027]).
With regard to claim 86, which recites “the PEgRNA further comprises an RNA-binding protein recruitment domain,” as set forth above, the retron-guide cassette of Fraser reads on the instantly claimed PEgRNA. Zhang further discloses that the guide molecule taught therein comprises an aptamer sequence capable of binding to an RNA-binding adaptor protein such as MS2 (paragraphs [0011], [0315], and [0316), which reads on the instantly claimed RNA-binding protein recruitment domain.
With regard to claim 87, which recites “the RNA-binding protein recruitment domain [of the method of claim 86] is an MS2 aptamer,” as set forth above, Zhang further discloses that the guide molecule taught therein comprises an aptamer sequence capable of binding to an RNA-binding adaptor protein such as MS2 (paragraphs [0011], [0315], and [0316).
With regard to claim 88, which recites “the polymerase [of the method of claim 85] is fused to an RNA-binding protein domain,” Zhang further discloses that the adenosine deaminase effector taught therein may be fused to RNA-binding protein MS2 coat protein (paragraphs [0315] and [0316]). While the adenosine deaminase effector of Zhang does not read on the instantly claimed polymerase, the disclosure of Zhang is applied to establish that principles of coordinating activity of a Cas protein and an editing enzyme, as in the instant application, Fraser, and Komor. Therefore, Zhang teaches that the effector/editing enzyme taught therein may be fused to an RNA-binding protein such as MS2 coat protein for purposes of implementing orthogonal gene editing (paragraphs [0315], [0316], and [0325]).
With regard to claim 89, which recites “the polymerase [of the method of claim 86] is fused to an MS2cp domain,” as set forth above, Zhang further discloses that the adenosine deaminase effector taught therein may be fused to RNA-binding protein MS2 coat protein for purposes of implementing orthogonal gene editing (paragraphs [0315], [0316], and [0325]).
With regard to claim 90, which recites “the double stranded DNA [of the method of claim 1] is in a cell,” Fraser discloses that the methods taught therein may be practiced within a genome of a host cell, wherein the host cell is transformed with a vector carrying the machinery set forth above and is subsequently edited (paragraph [0024]).
With regard to claim 91, which recites “the method of claim 90, wherein the cell is in a human,” this recitation is not patentably distinct from copending claims 9-11, which recite a method of prime editing in a human cell.
With regard to claim 92, which recites “the method of claim 90, further comprising delivering an mRNA encoding the prime editor into the cell,” as set forth above, Fraser and Komor collectively disclose the instantly claimed prime editor. While Fraser discloses that the reverse transcriptase and Cas effector taught therein may be expressed from a vector or plasmid (paragraphs [0026], [0027], and [0145]), Fraser does not explicitly disclose that these components are delivered as an mRNA. This deficiency is cured by Zhang, which discloses that the editing compositions disclosed therein (including Cas-enzyme fusions) may be delivered as mRNA molecules encoding all of the components thereof (paragraphs [0318] and [0319]).
With regard to claim 93, which recites “the mRNA and the pegRNA [of the method of claim 92] are delivered via a lipid nanoparticle,” Zhang further discloses that the mRNA encoding the components of the system taught therein (and set forth above) may be delivered via one or more lipid nanoparticles (paragraphs [0318] and [0319]).
With regard to claim 94, which recites “the method of claim 1, further comprising contacting the double stranded DNA with a complex comprising the prime editor and a nicking guide RNA (ngRNA),” Fraser further discloses that providing at least two DNA-targeting RNAs targeting opposite DNA strands facilitates a double-nicked double-strand break that favors HDR and decreases the frequency of insertion/deletion mutations at off-target DNA sites (paragraph [0143]). Per Fraser, two nickases of the invention disclosed therein may be utilized to create two single-stranded breaks on opposite strands of a target DNA, as set forth above (paragraph [0066]).
With regard to claim 95, which recites “the ngRNA [of the method of claim 94] comprises a[n] ngRNA spacer sequence capable of targeting the napDNAbp to the non-target strand of the double-stranded DNA sequence,” as set forth above, Fraser further discloses that providing at least two DNA-targeting RNAs targeting opposite DNA strands facilitates a double-nicked double-strand break that favors HDR and decreases the frequency of insertion/deletion mutations at off-target DNA sites (paragraph [0143]). Per Fraser, two nickases of the invention disclosed therein may be utilized to create two single-stranded breaks on opposite strands of a target DNA (paragraph [0066]). The DNA-targeting RNAs of Fraser read on the guide RNAs of Fraser, which are disclosed to comprise a crRNA spacer sequence that is complementary to a target nucleic acid sequence (paragraph [0127]).
Given that copending application ‘899 also discloses PEgRNAs, as in the instant application, that Fraser and Komor collectively disclose a method of editing a target genome utilizing said PEgRNAs, that Huang and UniProt disclose the napDNAbp and MMLV-RT sequences of the instant and patented applications, and that Zhang discloses the utility of including an MS2 coat protein and associated binding aptamer thereof in such systems, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to utilize the PEgRNA of copending application ‘899 in the systems collectively disclosed by the prior art, as well as to include an MS2 coat protein and associated binding aptamer thereof to predictably prime edit a double-stranded DNA, as instantly claimed. One would have been motivated to make such a modification in order to receive the expected benefit of practicing prime editing.
This is a provisional nonstatutory double patenting rejection.
Conclusion
No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Sarah E Allen whose telephone number is (571)272-0408. The examiner can normally be reached M-F 8-5.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Jennifer Dunston can be reached at 571-272-2916. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/SARAH E ALLEN/ Examiner, Art Unit 1637
/J. E. ANGELL/ Primary Examiner, Art Unit 1637
Prosecution Insights