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 .
Claims 1-11, 13-19, 21, and 25 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.
Based on the disclosure of provisional application 63/140,032, instant claims 1-4, 9-11, 13-19, 21, and 25 have an effective filing date of 01/21/2021, while instant claims 5-8 have an effective filing date of 01/21/2022, which is the filing date of the associated international application PCT/US2022/013390.
Information Disclosure Statement
Receipt of information disclosure statements on 01/15/2025 and 09/09/2025 is acknowledged. The signed and initialed PTO-1449‘s have been mailed with this action.
Drawings
The drawings are objected to because:
37 CFR 1.84 (u)(1) states “View numbers must be preceded by the abbreviation "FIG."” In the current case, the view numbers for Figures 1-17 are preceded by the word "Figure" instead of the abbreviation "FIG.". It would be remedial to replace “Figure” with the abbreviation “FIG.”.
With specific regard to Figures 1B and 1C, both Figures 1B and 1C disclose “3 Gene KO” and “4 Gene KO.” While the associated brief description of the drawing in the instant specification discloses that genes TRAC, PDl, B2M, and CISH were knocked out from primary human T cells using the multienzyme system taught therein, this disclosure only clarifies the 4 gene knockout. It is not clear from the drawing itself or the associated description what the disclosed “3 Gene KO” signifies. Furthermore, in Figure 1B specifically, both the 3 Gene KO and 4 Gene KO bars are visually indistinguishable from one another. For purposes of clarity, it would be remedial to clarify what “3 Gene KO” signifies in Figures 1B and 1C, as well as to visually distinguish the 3 Gene KO and 4 Gene KO bars of Figure 1B.
With specific regard to Figures 3-5, the image quality is insufficient to be clearly legible/interpretable. It would be remedial to increase the image quality such that it is clearly legible/interpretable.
With specific regard to Figures 7A and 7B, the key of the depicted bar graph indicates that the various bars correspond to Pulse, Nickase Cas9, ABE8e, and ABE8e Retargeting. However, the corresponding bars are visually indistinguishable from one another. For purposes of clarity, it would be remedial to visually distinguish the various bars from one another.
With specific regard to Figure 13, which includes sub-figures 13A-13N, the associated brief description of the drawings (page 5, line 30-page 6, line 15) is not structured such that it clearly indicates what each sub-figure is depicting. For example, it does not appear that sub-figure (M) has any associated description in the instant specification. It would be remedial to clarify the subject matter disclosed in Figure 13 in the associated brief description, as set forth below in the section addressing objections to the Specification.
Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
Specification
The disclosure is objected to because of the following informalities:
As set forth above regarding Figure 13, which includes sub-figures 13A-13N, the associated brief description of the drawings (page 5, line 30-page 6, line 15) is not structured such that it clearly indicates what each sub-figure is depicting. For example, it does not appear that sub-figure (M) has any associated description in the instant specification. Furthermore, the structure of the description of Figure 13 is not clear, especially regarding the intermixed Figure labels and abbreviations. These labels are not immediately distinguishable from each other, and the description is not structured such that it clarifies these labels. It would be remedial to clearly address each sub-figure and define all abbreviations such that they are clearly distinguished from the sub-figure labels.
With regard to Table 2, all columns of said Table are populated through SEQ ID NO: 21. However, the entries for SEQ ID NOs: 22-25 are only populated in the “Gene,” “gRNA name,” and “5’-gRNA Sequence-3’” columns. They are not populated in the “Orientation,” “Target base(s),” or “Predicted Outcome” columns. It is not immediately clear why these columns are not populated. It would be remedial to ensure Table 2 is fully populated, or to clearly address the significance of why certain columns are not populated.
Appropriate correction is required.
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 1-3, 5-9, and 11 are objected to because of the following informalities:
Claims 1, 2, 6, 7, and 9 all recite introduction of “gRNAs,” which is an acronymization of “guide RNA” per the instant specification (page 9, lines 23-24). Prior to reciting an acronym, it is proper to first recite the term being acronymized. In the instant case, it would be remedial to amend the first recitation of “gRNAs” to recite “guide RNAs (gRNAs)” to properly establish the basis of the acronym used throughout the instant claim set.
Claims 1-3 and 5-9 all recite the term “knockin,” which is not consistent with how this term is conventionally written in the field. As disclosed in WO 2019/178225 A2 (hereinafter Moriarity; as cited in the IDS filed 01/15/2025), the introduction of a specified donor sequence into a targeted genomic locus is conventionally written as “knock-in” (bolded and underlined emphasis added). In order to comport with conventions in the field, it would be remedial to recite “knock-in” in place of “knockin” in the instant claim set.
Claims 1, 2, 6, and 7 are objected to for reciting including periods in the recitation of the method steps claimed therein. According to MPEP 608.01(m), “Each claim begins with a capital letter and ends with a period. Periods may not be used elsewhere in the claims except for abbreviations. See Fressola v. Manbeck, 36 USPQ2d 1211 (D.D.C. 1995)”. It would be remedial to replace the periods of instant claims 1, 2, 6, and 7 with appropriate punctuation, such as parentheses.
Claim 11 does not comply with standard grammatical and/or linguistic conventions. The preamble “the method of claim 10” is not properly followed by a comma separating the preamble from the body of the claim. It would be remedial to amend instant claim 11 such that it comports with standard grammatical and/or linguistic conventions, for example by reciting “the method of claim 10, wherein the knockout…” (bolded and underlined emphasis added).
Appropriate correction is required.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Section 33(a) of the America Invents Act reads as follows:
Notwithstanding any other provision of law, no patent may issue on a claim directed to or encompassing a human organism.
Claim 25 is rejected under 35 U.S.C. 101 and section 33(a) of the America Invents Act as being directed to or encompassing a human organism. See also Animals - Patentability, 1077 Off. Gaz. Pat. Office 24 (April 21, 1987) (indicating that human organisms are excluded from the scope of patentable subject matter under 35 U.S.C. 101).
With regard to claim 25, which recites “a genetically modified cell obtained according to the method of claim 1,” the broadest reasonable interpretation of the term “cell” embraces a human having the cell. Neither the instant claim language nor the disclosure of the instant specification precludes this interpretation. It would be remedial to amend the instant claim to recite “an isolated genetically modified cell” to avoid the claim embracing a human organism.
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 2, 7, and 9-11 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 2 and 7 are both drawn to methods for producing genetically modified cells, wherein the methods further comprise “introducing into the cells at least one additional set of reagents that target a different knock[-]in target site.” The rejected claims thus comprise a set of additional reagents that are defined only by their ability to target a different knock-in target site. Thus, under broadest reasonable interpretation, the claims necessarily encompass any additional reagent capable of targeting a different knock-in target site.
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 targeting a different knock-in target site with an additional set of guide RNAs (page 14, lines 24-30). No description is provided of any other set of additional reagents that are able to target a different knock-in target site.
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 an additional set of guide RNAs. The results are not necessarily predictive of targeting a different knock-in target site with any other reagent capable of targeting a specific genomic site, such as a zinc finger protein or a TALEN (reviewed in Gaj et al., 2013), which have entirely different functional requirements and mechanisms of action than the disclosed CRISPR guide RNAs. Thus, it is impossible for one to extrapolate from the few examples described herein those sets of additional reagents that target a different knock-in target site 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 additional reagents that target a different knock-in target site.
Therefore, the skilled artisan would have reasonably concluded applicants were not in possession of the claimed invention for claims 2 and 7.
Claims 9-11 are drawn to a method of producing genetically modified cells, wherein the method further comprises introducing one or more primary gRNAs into the cells to generate genetically modified cells with at least one targeted knock-in and at least one targeted knockout. The rejected claims thus encompass introducing up to an unlimited number of gRNAs to produce genetically modified cells with up to an unlimited number of targeted knock-ins and up to an unlimited number of targeted knockouts. Claims 10 and 11 depend from claim 9 and do not limit the number of gRNAs, targeted knock-ins, or targeted knockouts introduced by the claimed method.
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 introduction of two nicking gRNAs (page 4, line 5) and four retargeting gRNAs (page 6, lines 14 and 27), as well as simultaneous knock-in of one gene and knockout of four genes (page 14, lines 6-10). No description is provided of introducing up to an unlimited number of gRNAs to produce genetically modified cells with up to an unlimited number of targeted knock-ins and up to an unlimited number of targeted knockouts, as is encompassed by the instant claim language.
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 a limited number of introduced gRNAs to induce a limited number of targeted knock-ins and knockouts. The results are not necessarily predictive of a method of producing genetically modified cells, wherein up to an unlimited number of gRNAs are introduced to induce up to an unlimited number of targeted knock-ins and knockouts. Thus, it is impossible for one to extrapolate from the few examples described herein those methods of producing genetically modified cells 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 a method of producing genetically modified cells, wherein up to an unlimited number of gRNAs are introduced to induce up to an unlimited number of targeted knock-ins and knockouts.
Therefore, the skilled artisan would have reasonably concluded applicants were not in possession of the claimed invention for claims 9-11.
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1-11, 13-19, 21, and 25 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
With regard to claims 1 (from which claims 2-5 and 9-11, 13-19, and 21 depend), 6 (from which claims 7 and 8 depend, and 25 (which directly invokes the subject matter of claim 1), each of the independent claims set forth above recites or otherwise requires “a plasmid, mRNA, or protein encoding a base editor” (bolded emphasis added). As is known to those of ordinary skill in the art, both plasmids and mRNA carry nucleic acid sequences that encode protein products. However, the protein products do not encode themselves, as may be reasonably interpreted from the instant claim language. None of the dependent claims resolve the basis of the indefiniteness rejection set forth above. It would be remedial to amend the instant claim language to clearly designate which molecules may encode the claimed base editor and which molecules correspond to the claimed base editor.
With regard to claims 2 and 7, both claims recite the limitation "each additional set of gRNAs" in lines 2-3. There is insufficient antecedent basis for this limitation in the claim. While the term “at least one additional set of reagents” is recited in line 2, these reagents are not limited to the later-recited set of gRNAs and thus cannot be considered to establish sufficient antecedent basis. It would be remedial to ensure that there is sufficient antecedent basis for each claim term.
Regarding claims 5 and 6, the phrase "preferably" renders the claim indefinite because it is unclear whether the limitation(s) following the phrase are part of the claimed invention. It would be remedial to clearly designate the bounds such that one of ordinary skill in the art would be reasonably apprised of the metes and bounds of protection sought by the instant claim language. See MPEP § 2173.05(d).
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, 13-19, 21 and 25 are rejected under 35 U.S.C. 103 as being unpatentable over WO 2019/178225 A2 (hereinafter Moriarity; as cited in the IDS filed 01/15/2025) in view of Liu et al., 2019 (hereinafter Liu), Singh et al., 2015 (hereinafter Singh), and Richter et al., 2020 (hereinafter Richter; as cited in the IDS filed 01/15/2025), as evidenced by Choi et al., 2021 (hereinafter Choi) and Plum et al., 1994 (hereinafter Plum).
With regard to claim 1, which recites “a method for producing genetically modified cells, the method comprising introducing into the cells:
i. a plasmid, mRNA, or protein encoding a base editor;
ii. two primary gRNAs that are complementary to the 5’ and 3’ ends of a knock[-]in target site prior to editing;
iii. two retargeting gRNAs that are complementary to the 5’ and 3’ ends of a knock[-]in target site after they have been edited by complexes comprising the base editor and the primary gRNAs; and
iv. a DNA donor template;
thereby generating cells with a targeted knock[-]in via homology directed repair from the DNA donor template,” Moriarity discloses methods and systems for generating multiplexed genetically modified cells via targeted gene disruption (i.e. gene knockout) and targeted gene knock-in of multiple genes accomplished using base editors and two guide RNAs, wherein said base editors are encoded in and provided via a plasmid or an mRNA and convert base pairs, for example such as A[Wingdings font/0x9F]T to G[Wingdings font/0x9F]C (abstract; paragraphs [0004], [0030], [0032], [0039], and [0040]). Furthermore, the knock-ins disclosed in Moriarity are generated by providing a DNA donor template to facilitate homology directed repair (HDR) (paragraphs [0004] and [0039]). Thus, Moriarity discloses that base editors can be used to generate multiplexed genetically modified cells, wherein multiple genes in said genetically modified cells are either knocked out or engineered via targeted knock in with a reduced rate of indel formation (paragraph [0030]). Regarding the gRNAs disclosed in Moriarity, while Moriarity does disclose two guide RNAs, they do not explicitly address that the gRNAs are complementary to the 5’ and 3’ ends of the target site, as is instantly claimed. This deficiency is cured by Choi, which discloses a method of prime editing that also simultaneously performs knock-in and knockout (Figure 2) that may be multiplexed (Figure 5). As shown in Figure 2, this system requires a pair of gRNAs (as in Moriarity) that targets the 5’ and 3’ ends of the target site, as is instantly claimed. Additionally, Moriarity does not disclose the instantly claimed retargeting gRNAs. The gRNAs of Moriarity are considered to read on the instantly claimed primary gRNAs, however, as the two guide RNAs disclosed in Moriarity must necessarily be complementary to the target site prior to editing (i.e. primary gRNAs), as they would otherwise be unable to facilitate the editing disclosed therein.
This deficiency is cured by Liu and Singh. As is known to those of ordinary skill in the art, each time a nuclease (such as a CRISPR nuclease) introduces a targeted break (or nick) in the genome, the break may be resolved either by non-homologous end joining (NHEJ) or HDR, with NHEJ occurring at higher efficiency than HDR (abstract). It is thus of substantial interest in the field to increase the efficiency of HDR, by methods such as inhibiting NHEJ or enhancing HDR by chemical modulation, synchronized expression, overlapping homology arms, and utilizing modified Cas9 nucleases, including base editors, which nick the targeted DNA rather than introducing more potently mutagenic double-strand breaks, thereby facilitating precision editing with a low rate of indels (abstract; section “ENHANCING HDR BY USING MODIFIED CAS9”). While these methods have somewhat increased the efficiency of HDR, the field is still actively working to continue increasing the efficiency of HDR (page 6, column 2, paragraph 2).
Another complicating factor in performing CRISPR-mediated HDR is the potential for continued recutting triggering mutagenic NHEJ following HDR, as if the edited locus does not comprise an edit that disrupts the targeted gRNA site or its associated PAM site, the site will continue to be targeted for cleavage, and at each cleavage event, either NHEJ (which occurs at higher rates) or HDR (which occurs at lower rates) will be triggered to repair the damage (reviewed in Singh: see section “Combating CRISPR Robustness and NHEJ”). Therefore, by extension, if the original HDR event introduces an edit that does disrupt the targeted gRNA site or its associated PAM site, that edit will prevent further targeting and cleavage (reviewed in Singh: see section “Combating CRISPR Robustness and NHEJ”).
Based on these disclosures, it would have been obvious to someone of ordinary skill in the art not only to harness the power of base editors for multiplexed gene editing comprising both knock-in and knockout events with a reduced rate of indel formation, as disclosed in Moriarity, but also to increase the rate of HDR based on the established principles in the field disclosed in Liu and Singh. Liu teaches that each cleavage event induced by a CRISPR enzyme (including base editors) will be resolved via NHEJ or HDR, wherein NHEJ typically occurs at a higher rate than HDR, although base editors have been shown to increase the rate of HDR following said cleavage events. Therefore, one of ordinary skill in the art would reasonably conclude that multiple cleavage events at the same site would provide multiple opportunities to initiate either NHEJ or HDR, with cleavage events introduced by base editors having a higher probability of resolving via HDR, thereby motivating multiple rounds of base editor-mediated cleavage, particularly in view of the known reduced rate of indel formation associated with such base editors.
Therefore, based on these disclosures, one of ordinary skill in the art would reasonably be motivated to repeatedly contact a site intended for knock-in with a base editor and a DNA donor template. However, Singh teaches that effective knock-in depends on preventing induction of NHEJ at the knocked-in site by disrupting the complementarity between provided gRNAs and the target sites associated with the same. Thus, one of ordinary skill in the art would be aware that a system to increase the rate of HDR, wherein the rate of HDR is increased by repeatedly cleaving a target site with a base editor to induce HDR, must also block NHEJ from disrupting the engineered knock-in. As disclosed in Moriarity, base editors convert base pairs (i.e. A[Wingdings font/0x9F]T to G[Wingdings font/0x9F]C) and thus, successful editing with a base editor must necessarily disrupt the complementarity between the originally-provided gRNAs (i.e. the instantly claimed primary gRNAs). However, this disruption blocks further editing, as taught in Singh. Accordingly, if knock-in did not occur during the original editing event, no further knock-in could be facilitated by targeting the base editor with the originally-provided gRNAs (i.e. the instantly claimed primary gRNAs). Therefore, a new pair of gRNAs (i.e. the instantly claimed retargeting gRNAs) would be required to target the base-edited locus for successful knock-in. Conversely, if knock-in did occur during the original editing event, one of ordinary skill in the art would have been motivated to engineer the DNA donor template disclosed in Moriarity to otherwise disrupt gRNA targeting to the desired locus, for example by incorporating a mutated PAM site, as taught in Singh. Thus, if successful knock-in is achieved in the first round of editing following targeting with primary gRNAs, predictably no further editing events will be initiated, and if successful knock-in is not achieved in the first round of editing following targeting with primary gRNAs, the retargeting gRNAs will predictably continue targeting the designated site until knock-in is successfully achieved or the process is otherwise halted.
Thus, it is considered that Moriarity, Choi, Liu, and Singh collectively motivate and disclose the method of instant claim 1.
With regard to claim 2, which recites “the method [of claim 1] further comprises introducing into the cells at least one additional set of reagents that target a different knock[-]in target site, each additional set of gRNAs comprising:
i. two primary gRNAs that are complementary to two knock[-]in target sites prior to editing;
ii. two retargeting gRNAs that are complementary to the two knock[-]in target sites after they have been edited by complexes comprising the base editor and the primary gRNAs; and
iii. a DNA donor template;
wherein the method generates cells with two or more targeted knock[-]ins,” as set forth above, Moriarity, Choi, Liu, and Singh collectively motivate and disclose the method of instant claim 1. Moriarity specifically discloses that the methods of using base editors taught therein and set forth above are amenable to multiplexed disruption of multiple genes relevant to immunotherapy in cells, with or without knock-in of a target DNA template or missense mutation (paragraph [0040]). As disclosed in Choi, such multiplexed editing requires targeting multiple (i.e. two or more) target sites across multiple genes (Figure 5). Therefore, one of ordinary skill in the art would be motivated to apply the method comprising the instantly claimed primary and re-targeting gRNAs set forth above (motivated and disclosed by Moriarity, Choi, Liu, and Singh) to multiple target sites (i.e. two or more), as disclosed in Choi, to predictably generate genetically modified cells with multiplexed edits, as disclosed in Moriarity.
Thus, it is considered that Moriarity, Choi, Liu, and Singh collectively motivate and disclose the method of instant claim 2.
With regard to claims 3-5, which respectively recite “the method [of claim 1] generates cells with a targeted knock[-]in with at least 40% efficiency,” “a targeted knockout with at least 90% efficiency,” and “the rate of indel formation at the knockin target site is less than 10%,” as set forth above, Moriarity, Choi, Liu, and Singh collectively motivate and disclose the method of instant claim 1. Therefore, the methods of both instant claim 1 and of Moriarity, Choi, Liu, and Singh must be structurally identical and therefore must necessarily have identical functions. See MPEP § 2114 and § 2173.05(g).
Thus, it is considered that Moriarity, Choi, Liu, and Singh collectively disclose the methods of claims 3-5.
With regard to claim 6, which recites “a method for producing genetically modified cells, the method comprising introducing into the cells:
i. a plasmid, mRNA, or protein encoding a base editor;
ii. at least two primary gRNAs that are complementary to the 5’ and 3’ ends of a knock[-] in target site prior to editing; and
iii. a DNA donor template;
thereby generating cells with a targeted knock[-]in via homology directed repair from the DNA donor template; and wherein the rate of indel formation at the knock[-]in target site is less than 10%, and is preferably less than 1%, optionally undetectable,” as set forth above, Moriarity discloses methods and systems for generating multiplexed genetically modified cells via targeted gene disruption (i.e. gene knockout) and targeted gene knock-in of multiple genes accomplished using base editors and two guide RNAs, wherein said base editors are encoded in and provided via a plasmid or an mRNA and convert base pairs, for example such as A[Wingdings font/0x9F]T to G[Wingdings font/0x9F]C (abstract; paragraphs [0004], [0030], [0032], [0039], and [0040]). Furthermore, the knock-ins disclosed in Moriarity are generated by providing a DNA donor template to facilitate homology directed repair (HDR) (paragraphs [0004] and [0039]). Thus, Moriarity discloses that base editors can be used to generate multiplexed genetically modified cells, wherein multiple genes in said genetically modified cells are either knocked out or engineered via targeted knock in with a reduced rate of indel formation (paragraph [0030]). Regarding the gRNAs disclosed in Moriarity, while Moriarity does disclose two guide RNAs, they do not explicitly address that the gRNAs are complementary to the 5’ and 3’ ends of the target site, as is instantly claimed. This deficiency is cured by Choi, which discloses a method of prime editing that also simultaneously performs knock-in and knockout (Figure 2) that may be multiplexed (Figure 5). As shown in Figure 2, this system requires a pair of gRNAs (as in Moriarity) that targets the 5’ and 3’ ends of the target site, as is instantly claimed. Furthermore, the two guide RNAs disclosed in Moriarity must necessarily be complementary to the target site prior to editing (i.e. primary gRNAs), as they would otherwise be unable to facilitate the editing disclosed in Moriarity.
Regarding the claimed rate of indel formation, it is considered that Moriarity and Choi collectively disclose the method of claim 6. Therefore, the methods of both instant claim 6 and of Moriarity and Choi must be structurally identical and therefore must necessarily have identical functions. See MPEP § 2114 and § 2173.05(g).
With regard to claim 7, which recites “the method [of claim 6] further comprises introducing into the cells at least one additional set of reagents that target a different knock[-]in target site, each additional set of gRNAs comprising:
i. two primary gRNAs that are complementary to two knock[-]in target sites prior to editing; and
ii. a DNA donor template; and
wherein the method generates cells with two or more targeted knock[-]ins,” as set forth above, Moriarity and Choi, Liu, and Singh collectively motivate and disclose the method of instant claim 6. Moriarity specifically discloses that the methods of using base editors taught therein and set forth above are amenable to multiplexed disruption of multiple genes relevant to immunotherapy in cells, with or without knock-in of a target DNA template or missense mutation (paragraph [0040]). As disclosed in Choi, such multiplexed editing requires targeting multiple (i.e. two or more) target sites across multiple genes (Figure 5). Therefore, one of ordinary skill in the art would be motivated to apply the method comprising the instantly claimed primary gRNAs set forth above (motivated and disclosed by Moriarity and Choi) to multiple target sites (i.e. two or more), as disclosed in Choi, to predictably generate genetically modified cells with multiplexed edits, as disclosed in Moriarity.
Thus, it is considered that Moriarity and Choi collectively motivate and disclose the method of instant claim 7.
With regard to claim 8, which recites “the method [of claim 6] generates cells with a targeted knock[-]in with at least 20% efficiency,” as set forth above, Moriarity and Choi collectively motivate and disclose the method of instant claim 6. Therefore, the methods of both instant claim 6 and of Moriarity and Choi must be structurally identical and therefore must necessarily have identical functions. See MPEP § 2114 and § 2173.05(g).
Thus, it is considered that Moriarity, Choi, Liu, and Singh collectively disclose the method of claim 8.
With regard to claim 9, which recites “the method [of claim 1] further comprises introducing into the cells: one or more primary gRNAs that are complementary to a knockout target site prior to editing, and wherein the method generates cells with at least one targeted knock[-]in and at least one targeted knockout,” as set forth above, Moriarity discloses a base-editor-facilitated multiplexed gene editing system for generating edited cells (specifically lymphohematopoietic cells) with both gene knockouts and knock-ins (abstract; paragraphs [0004], [0030], [0032], [0039], and [0040]). This system comprises administration of one or more gRNAs designed to produce one or more targeted knock-ins in lymphohematopoietic cells comprising at least one gene knockout (paragraph [0003]). While Moriarity does not explicitly disclose the administration of one or more gRNAs for gene knockout, it is well-known to those of ordinary skill in the art that gRNAs are required to target a site both for knock-in and for knockout with base editors and other CRISPR-based enzymes (reviewed in Singh: see section “A basic knockout”). Therefore, one of ordinary skill in the art would have reasonably been motivated to administer one or more gRNAs as per the disclosure of Moriarity to generate an edited cell comprising at least one gene knockout, as is also disclosure in Moriarity.
Thus, it is considered that Moriarity and Singh collectively motivate and disclose the additional limitations of instant claim 9.
With regard to claims 10 and 11, which respectively recite “the targeted knockout site [of the method of claim 9] is in a gene that encodes an alloreactive or immunomodulatory protein,” wherein said “gene is selected from TRAC, PDCD1, B2M, and CISH,” as set forth above, Moriarity discloses a base-editor-facilitated multiplexed gene editing system for generating edited cells (specifically lymphohematopoietic cells) with both gene knockouts and knock-ins (abstract; paragraphs [0004], [0030], [0032], [0039], and [0040]). Moriarity further discloses that the genetically edited cells produced therein include knockout of TRAC (paragraph [0009]). Per the instant specification, the TRAC gene is an alloreactive or immunomodulatory gene (page 19, lines 2-17). Additionally, TRAC is explicitly recited at instant claim 11.
Thus, it is considered that Moriarity discloses the additional limitations of instant claims 10 and 11.
With regard to claims 13 and 14, which respectively recite “the base editor [of the method of claim 1] is BE3, BE4, or ABE8e,” or is limited to only “ABE8e,” as set forth above, Moriarity discloses a base-editor-facilitated multiplexed gene editing system for generating edited cells (specifically lymphohematopoietic cells) with both gene knockouts and knock-ins (abstract; paragraphs [0004], [0030], [0032], [0039], and [0040]). While Moriarity discloses that base editors BE3, BE4, and ABE may be used in the system taught therein (paragraph [0041]), they do not disclose base editor ABE8e, as is instantly claimed. This deficiency is cured by Richter, which discloses the development of base editor ABE8e (abstract). Richter further discloses that ABE8e is particularly well-suited to multiplex editing applications (such as the multiplex editing disclosed in Moriarity), has an expanded targeting scope, and performs more efficiently (abstract; page 890, column 1, paragraph 4).
Thus, it is considered that Moriarity and Richter collectively disclose the additional limitations of instant claims 13 and 14.
With regard to claims 15-17, which respectively recite that “the cells [of the method of claim 1] are lymphohematopoietic cells,” including “T cells or natural killer (NK) cells,” and “pluripotent stem cells or progenitor cells capable of differentiating into T cells or NK cells,” as set forth above, Moriarity discloses a base-editor-facilitated multiplexed gene editing system for generating edited cells (specifically lymphohematopoietic cells) with both gene knockouts and knock-ins (abstract; paragraphs [0004], [0030], [0032], [0039], and [0040]). The edited lymphohematopoietic cells disclosed in Moriarity are further disclosed to be T cells or NK cells, as well as CD34+ hematopoietic stem progenitor cells (paragraph [0003]). As is known to those of ordinary skill in the art, CD34+ hematopoietic stem progenitor cells are capable of differentiating into mature T cells (taught in Plum: see abstract), as instantly claimed.
Thus, it is considered that Moriarity discloses the additional limitations of instant claims 15-17, as evidenced by Plum.
With regard to claim 18, which recites “the DNA donor template [of the method of claim 1] is provided via a recombinant adeno-associated virus (rAAV),” as set forth above, Moriarity discloses a multiplexed gene editing system for generating edited lymphohematopoietic cells with both gene knockouts and knock-ins, wherein said knock-ins are generated by providing a DNA donor template (abstract; paragraphs [0004], [0030], [0032], [0039], and [0040]). Furthermore, the knock-ins disclosed in Moriarity are generated by providing a DNA donor template to facilitate homology directed repair (HDR) (paragraphs [0004] and [0039]). Said DNA donor is delivered via rAAV per paragraph [0039]).
Thus, it is considered that Moriarity discloses the additional limitations of instant claim 18.
With regard to claim 19, which recites “the DNA donor template [of the method of claim 1] encodes a chimeric antigen receptor (CAR),” as set forth above, Moriarity discloses a multiplexed gene editing system for generating edited lymphohematopoietic cells with both gene knockouts and knock-ins, wherein said knock-ins are generated by providing a DNA donor template (abstract; paragraphs [0004], [0030], [0032], [0039], and [0040]). The DNA donor template of Moriarity is disclosed to encode a T cell receptor (TCR) and a chimeric antigen receptor (CAR) (paragraph [0004]).
Thus, it is considered that Moriarity discloses the additional limitations of instant claim 19.
With regard to claim 21, which recites “the DNA donor template [of the method of claim 1] encodes a T cell receptor (TCR),” as set forth above, Moriarity discloses a multiplexed gene editing system for generating edited lymphohematopoietic cells with both gene knockouts and knock-ins, wherein said knock-ins are generated by providing a DNA donor template (abstract; paragraphs [0004], [0030], [0032], [0039], and [0040]). The DNA donor template of Moriarity is disclosed to encode a T cell receptor (TCR) and a chimeric antigen receptor (CAR) (paragraph [0004]).
Thus, it is considered that Moriarity discloses the additional limitations of instant claim 21.
With regard to claim 25, which recites “a genetically modified cell obtained according to the method of claim 1,” as set forth above, Moriarity discloses a base-editor-facilitated multiplexed gene editing system for generating edited cells (specifically lymphohematopoietic cells) with both gene knockouts and knock-ins (abstract; paragraphs [0004], [0030], [0032], [0039], and [0040]). Moriarity further discloses a genetically modified cell obtained according to the methods taught therein (paragraph [0005]).
Thus, it is considered that Moriarity discloses the additional limitations of instant claim 25.
Given that Moriarity discloses a base-editor-facilitated multiplexed gene editing system for generating edited cells comprising both gene knockouts and knock-ins (as set forth above); that Liu discloses that cleavage by CRISPR nucleases may be resolved by either NHEJ (i.e. knockout), which occurs at higher rates, or HDR (i.e. knock-in), which occurs at lower rates, and that base editors (such as ABE8e, which is well-suited for multiplexed gene editing per Richter) enhance knock-in facilitated by HDR and reduce the rate of indels introduced by said editing machinery; and that Singh discloses that unless the knocked-in sequence introduces an edit that disrupts the targeted gRNA site or its associated PAM site, the knocked-in site will be targeted in further rounds of editing and thus may be subjected to mutagenic NHEJ (or HDR) following each cleavage event, 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 Moriarity to improve efficiency of the knock-in events of the multiplexed editing system disclosed therein such that the site intended for knock-in is repeatedly targeted with a base editor (such as ABE8e as disclosed in Richter) and a DNA donor template engineered to disrupt gRNA targeting to the desired locus (as disclosed in Singh), such that if successful knock-in is achieved in the first round of editing, predictably no further editing events will be initiated, and if successful knock-in is not achieved in the first round of editing, the provided retargeting gRNAs targeting the base-edited site will predictably continue targeting the designated site until knock-in is successfully achieved or the process is otherwise halted. One would have been motivated to make such a modification in order to receive the expected benefit of increasing rates of targeted knock-in in multiplexed gene editing facilitated by base editors.
Conclusion
No claims are allowed.
Claims 1-3, 5-9, and 11 are also objected to for the reasons indicated herein.
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/SARAH E ALLEN/ Examiner, Art Unit 1637
/J. E. ANGELL/ Primary Examiner, Art Unit 1637